Rationally Designed, Synthetic Antibody Libraries and Uses Therefor

ABSTRACT

The present invention overcomes the inadequacies inherent in the known methods for generating libraries of antibody-encoding polynucleotides by specifically designing the libraries with directed sequence and length diversity. The libraries are designed to reflect the preimmune repertoire naturally created by the human immune system, with or without DH segments derived from other species, and are based on rational design informed by examination of publicly available databases of antibody sequences.

RELATED APPLICATION

This application is a continuation-in-part of U.S. patent application Ser. No. 12/210,072 filed Sep. 12, 2008. This application also claims priority to U.S. provisional application Ser. No. 60/993,785, filed on Sep. 14, 2007, incorporated herein in its entirety by this reference.

BACKGROUND OF THE INVENTION

Antibodies have profound relevance as research tools and in diagnostic and therapeutic applications. However, the identification of useful antibodies is difficult and once identified, antibodies often require considerable redesign or ‘humanization’ before they are suitable for therapeutic applications.

Previous methods for identifying desirable antibodies have typically involved phage display of representative antibodies, for example human libraries derived by amplification of nucleic acids from B cells or tissues, or, alternatively, synthetic libraries. However, these approaches have limitations. For example, most human libraries known in the art contain only the antibody sequence diversity that can be experimentally captured or cloned from the source (e.g., B cells). Accordingly, the human library may completely lack or under-represent certain useful antibody sequences. Synthetic or consensus libraries known in the art have other limitations, such as the potential to encode non-naturally occurring (e.g., non-human) sequences that have the potential to be immunogenic. Moreover, certain synthetic libraries of the art suffer from at least one of two limitations: (1) the number of members that the library can theoretically contain (i.e., theoretical diversity) may be greater than the number of members that can actually be synthesized, and (2) the number of members actually synthesized may be so great as to preclude screening of each member in a physical realization of the library, thereby decreasing the probability that a library member with a particular property may be isolated.

For example, a physical realization of a library (e.g., yeast display, phage display, ribosomal display, etc.) capable of screening 10¹² library members will only sample about 10% of the sequences contained in a library with 10¹³ members. Given a median CDRH3 length of about 12.7 amino acids (Rock et al, J. Exp. Med., 1994, 179:323-328), the number of theoretical sequence variants in CDRH3 alone is about 20^(12.7), or about 3.3×10¹⁶ variants. This number does not account for known variation that occurs in CDRH1 and CDRH2, heavy chain framework regions, and pairing with different light chains, each of which also exhibit variation in their respective CDRL1, CDRL2, and CDRL3. Finally, the antibodies isolated from these libraries are often not amenable to rational affinity maturation techniques to improve the binding of the candidate molecule.

Accordingly, a need exists for smaller (i.e., able to be synthesized and physically realizable) antibody libraries with directed diversity that systematically represent candidate antibodies that are non-immunogenic (i.e., more human) and have desired properties (e.g., the ability to recognize a broad variety of antigens). However, obtaining such libraries requires balancing the competing objectives of restricting the sequence diversity represented in the library (to enable synthesis and physical realization, potentially with oversampling, while limiting the introduction of non-human sequences) while maintaining a level of diversity sufficient to recognize a broad variety of antigens. Prior to the instant invention, it was known in the art that “[al]though libraries containing heavy chain CDR3 length diversity have been reported, it is impossible to synthesize DNA encoding both the sequence and the length diversity found in natural heavy chain CDR3 repertoires” (Hoet et al., Nat. Biotechnol., 2005, 23: 344, incorporated by reference in its entirety).

Therefore, it would be desirable to have antibody libraries which (a) can be readily synthesized, (b) can be physically realized and, in certain cases, oversampled, (c) contain sufficient diversity to recognize all antigens recognized by the preimmune human repertoire (i.e., before negative selection), (d) are non-immunogenic in humans (i.e., comprise sequences of human origin), and (e) contain CDR length and sequence diversity, and framework diversity, representative of naturally-occurring human antibodies. Embodiments of the instant invention at least provide, for the first time, antibody libraries that have these desirable features.

SUMMARY OF THE INVENTION

The present invention is relates to, at least, synthetic polynucleotide libraries, methods of producing and using the libraries of the invention, kits and computer readable forms including the libraries of the invention. In some embodiments, the libraries of the invention are designed to reflect the preimmune repertoire naturally created by the human immune system and are based on rational design informed by examination of publicly available databases of human antibody sequences. It will be appreciated that certain non-limiting embodiments of the invention are described below. As described throughout the specification, the invention encompasses many other embodiments as well.

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode at least 10⁶ unique antibody CDRH3 amino acid sequences comprising:

-   -   (i) an N1 amino acid sequence of 0 to about 3 amino acids,         wherein each amino acid of the N1 amino acid sequence is among         the 12 most frequently occurring amino acids at the         corresponding position in N1 amino acid sequences of CDRH3 amino         acid sequences that are functionally expressed by B cells;     -   (ii) a non-human CDRH3 DH amino acid sequence, N- and C-terminal         truncations thereof, or a sequence of at least about 80%         identity to any of them;     -   (iii) an N2 amino acid sequence of 0 to about 3 amino acids,         wherein each amino acid of the N2 amino acid sequence is among         the 12 most frequently occurring amino acids at the         corresponding position in N2 amino acid sequences of CDRH3 amino         acid sequences that are functionally expressed by B cells; and     -   (iv) a human CDRH3 H3-JH amino acid sequence, N-terminal         truncations thereof, or a sequence of at least about 80%         identity to any of them.

In one aspect of the invention, the B cells are human B cells. In still other embodiments of the invention, the B cells are non-human B cells. In yet another embodiment of the invention, the B cells are vertebrate B cells.

In certain other embodiments, the invention provides that the non-human CDRH3 DH amino acid sequence is a sequence from a vertebrate species. In some embodiments, the vertebrate species is selected from the group consisting of Mus musculus, Camelus sp., Llama sp., Camelidae sp., Raja sp., Ginglymostoma sp., Carcharhinus sp., Heterodontus sp., Hydrolagus sp., Ictalurus sp., Gallus sp., Bos sp., Marmaronetta sp., Aythya sp., Netta sp., Equus sp., Pentalagus sp., Bunolagus sp., Nesolagus sp., Romerolagus sp., Brachylagus sp., Sylvilagus sp., Oryctolagus, sp., Poelagus sp., Ovis sp., Sus sp., Gadus sp., Salmo sp., Oncorhynchus sp, Macaca sp., Rattus sp., Pan sp., Hexanchus sp., Heptranchias sp., Notorynchus sp., Chlamydoselachus sp., Heterodontus sp. Pristiophorus sp., Pliotrema sp., Squatina sp., Carcharia sp., Mitsukurina sp., Lamma sp., Isurus sp., Carcharodon sp., Cetorhinus sp., Alopias sp., Nebrius sp., Stegostoma sp., Orectolobus sp., Eucrossorhinus sp., Sutorectus sp., Chiloscyllium sp., Hemiscyllium sp., Brachaelurus sp., Heteroscyllium sp., Cirrhoscyllium sp., Parascyllium sp., Rhincodon sp., Apristurus sp., Atelomycterus sp., Cephaloscyllium sp., Cephalurus sp., Dichichthys sp., Galeus sp., Halaelurus sp., Haploblepharus sp., Parmaturus sp., Pentanchus sp., Poroderna sp., Schroederichthys sp., Scyliorhinus sp., Pseudotriakis sp., Scylliogaleus sp., Furgaleus sp., Hemitriakis sp., Mustelus sp., Triakis sp., Iago sp., Galeorhinus sp., Hypogaleus sp., Chaenogaleus sp., Hemigaleus sp., Paragaleus sp., Galeocerdo sp., Prionace sp., Sciolodon sp., Loxodon sp., Rhizoprionodon sp., Aprionodon sp., Negaprion sp., Hypoprion sp., Carcharhinus sp., Isogomphodon sp., Triaenodon sp., Sphyrna sp., Echinorhinus sp., Oxynotus sp., Squalus sp., Centroscyllium sp., Etmopterus sp., Centrophorus sp., Cirrhigaleus sp., Deania sp., Centroscymnus sp., Scymnodon sp., Dalatias sp., Euprotomicrus sp., Isistius sp., Squaliolus sp., Heteroscymnoides sp., Somniosus sp. and Megachasma sp.

In other embodiments of the invention, an antibody may be isolated from the polypeptide expression products of any of the libraries described herein. In yet another embodiment, the CDRH3 amino acid sequences are expressed as part of full-length heavy chains. In one aspect of the invention, the polynucleotides further encode an alternative scaffold. In yet another embodiment, the library of polypeptides is encoded by the synthetic polynucleotide library described herein.

In certain embodiments of the invention, a library of vectors may comprise the polynucleotide library described herein. In still another embodiment, a population of cells may comprise the vectors described herein. In yet another embodiment, the cells are yeast cells (e.g., S. cerevisiae).

In yet another embodiment, the invention includes a method of preparing a synthetic polynucleotide library comprising providing the polynucleotide sequences of claim 1 and assembling said polynucleotide sequences in the order [N1]-[DH]-[N2]-[H3-JH]. In still another embodiment, the invention provides a method of isolating one or more host cells expressing one or more antibodies, the method comprising:

-   -   (i) expressing a polypeptide comprising a CDRH3 sequence of         claim 1 in one or more host cells;     -   (ii) contacting the host cells with one or more antigens; and     -   (iii) isolating one or more host cells having antibodies that         bind to the one or more antigens.

In certain aspects, the invention includes a kit comprising any of the libraries of synthetic polynucleotides described herein.

In other aspects of the invention, the CDRH3 amino acid sequences encoded by the libraries of synthetic polynucleotides described herein are in computer readable form.

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode at least 10⁶ unique antibody CDRH3 amino acid sequences comprising:

-   -   (i) an N1 amino acid sequence of 0 to about 3 amino acids,         wherein each amino acid of the N1 amino acid sequence is among         the 12 most frequently occurring amino acids at the         corresponding position in N1 amino acid sequences of CDRH3 amino         acid sequences that are functionally expressed by human B cells;     -   (ii) a human CDRH3 DH amino acid sequence, N- and C-terminal         truncations thereof, or a sequence of at least about 80%         identity to any of them;     -   (iii) an N2 amino acid sequence of 0 to about 3 amino acids,         wherein each amino acid of the N2 amino acid sequence is among         the 12 most frequently occurring amino acids at the         corresponding position in N2 amino acid sequences of CDRH3 amino         acid sequences that are functionally expressed by human B cells;         and     -   (iv) a human CDRH3H3-JH amino acid sequence, N-terminal         truncations thereof, or a sequence of at least about 80%         identity to any of them.

In other embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode at least about 10⁶ unique antibody CDRH3 amino acid sequences comprising:

-   -   (i) an N1 amino acid sequence of 0 to about 3 amino acids,         wherein:         -   (a) the most N-terminal N1 amino acid, if present, is             selected from a group consisting of R, G, P, L, S, A, V, K,             I, Q, T and D;         -   (b) the second most N-terminal N1 amino acid, if present, is             selected from a group consisting of G, P, R, S, L, V, E, A,             D, I, T and K; and         -   (c) the third most N-terminal N1 amino acid, if present, is             selected from the group consisting of G, R, P, S, L, A, V,             T, E, D, K and F;     -   (ii) a human CDRH3 DH amino acid sequence, N- and C-terminal         truncations thereof, or a sequence of at least about 80%         identity to any of them;     -   (iii) an N2 amino acid sequence of 0 to about 3 amino acids,         wherein:         -   (a) the most N-terminal N2 amino acid, if present, is             selected from a group consisting of G, P, R, L, S, A, T, V,             E, D, F and H;         -   (b) the second most N-terminal N2 amino acid, if present, is             selected from a group consisting of G, P, R, S, T, L, A, V,             E, Y, D and K; and         -   (c) the third most N-terminal N2 amino acid, if present, is             selected from the group consisting of G, P, S, R, L, A, T,             V, D, E, W and Q; and     -   (iv) a human CDRH3 H3-JH amino acid sequence, N-terminal         truncations thereof, or a sequence of at least about 80%         identity to any of them.

In still other embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode at least about 10⁶ unique antibody CDRH3 amino acid sequences that are at least about 80% identical to an amino acid sequence represented by the following formula:

[X]-[N1]-[DH]-[N2]-[H3-JH], wherein:

-   -   (i) X is any amino acid residue or no amino acid residue;     -   (ii) N1 is an amino acid sequence selected from the group         consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL,         GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV,         RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG,         PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT,         GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA,         EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL,         QT, PA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK,         SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE,         AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL,         SGL, SSE, TGL, WGT, and combinations thereof;     -   (iii) DH is an amino acid sequence selected from the group         consisting of all possible reading frames that do not include a         stop codon encoded by IGHD1-1, IGHD1-20, IGHD1-26, IGHD1-7,         IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16,         IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-17, IGHD4-23, IGHD4-4,         IGHD-4-11, IGHD5-12, IGHD5-24, IGHD5-5, IGHD-5-18, IGHD6-13,         IGHD6-19, IGHD6-25, IGHD6-6, and IGHD7-27, and N- and C-terminal         truncations thereof,     -   (iv) N2 is an amino acid sequence selected from the group         consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL,         GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV,         RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG,         PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT,         GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA,         EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL,         QT, PA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK,         SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE,         AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL,         SGL, SSE, TGL, WGT, and combinations thereof; and     -   (v) H3-JH is an amino acid sequence selected from the group         consisting of AEYFQH (SEQ ID NO: 17), EYFQH, YFQH, FQH, QH, H,         YWYFDL (SEQ ID NO: 18), WYFDL, YFDL, FDL, DL, L, AFDV (SEQ ID         NO: 19), FDV, DV, V, YFDY (SEQ ID NO: 20), FDY, DY, Y, NWFDS         (SEQ ID NO: 21), WFDS, FDS, DS, S, YYYYYGMDV (SEQ ID NO: 22),         YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, and MDV, or a sequence         of at least 80% identity to any of them.

In still another embodiment, the invention comprises wherein said library consists essentially of a plurality of polynucleotides encoding CDRH3 amino acid sequences that are at least about 80% identical to an amino acid sequence represented by the following formula:

[X]-[N1]-[DH]-[N2]-[H3-JH], wherein:

-   -   (i) X is any amino acid residue or no amino acid residue;     -   (ii) N1 is an amino acid sequence selected from the group         consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL,         GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV,         RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG,         PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT,         GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA,         EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL,         QT, PA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK,         SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE,         AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL,         SGL, SSE, TGL, WGT, and combinations thereof;     -   (iii) DH is an amino acid sequence selected from the group         consisting of all possible reading frames that do not include a         stop codon encoded by IGHD1-1, IGHD1-20, IGHD1-26, IGHD1-7,         IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16,         IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-17, IGHD4-23, IGHD4-4,         IGHD-4-11, IGHD5-12, IGHD5-24, IGHD5-5, IGHD-5-18, IGHD6-13,         IGHD6-19, IGHD6-25, IGHD6-6, and IGHD7-27, and N- and C-terminal         truncations thereof;     -   (iv) N2 is an amino acid sequence selected from the group         consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL,         GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV,         RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG,         PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT,         GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA,         EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL,         QT, PA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK,         SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE,         AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL,         SGL, SSE, TGL, WGT, and combinations thereof; and     -   (v) H3-JH is an amino acid sequence selected from the group         consisting of AEYFQH (SEQ ID NO: 17), EYFQH, YFQH, FQH, QH, H,         YWYFDL (SEQ ID NO: 18), WYFDL, YFDL, FDL, DL, L, AFDV (SEQ ID         NO: 19), FDV, DV, V, YFDY (SEQ ID NO: 20), FDY, DY, Y, NWFDS         (SEQ ID NO: 21), WFDS, FDS, DS, S, YYYYYGMDV (SEQ ID NO: 22),         YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, and MDV, or a sequence         of at least 80% identity to any of them.

In another embodiment, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode one or more full length antibody heavy chain sequences, and wherein the CDRH3 amino acid sequences of the heavy chain comprise:

-   -   (i) an N1 amino acid sequence of 0 to about 3 amino acids,         wherein each amino acid of the N1 amino acid sequence is among         the 12 most frequently occurring amino acids at the         corresponding position in N1 amino acid sequences of CDRH3 amino         acid sequences that are functionally expressed by human B cells;     -   (ii) a human CDRH3 DH amino acid sequence, N- and C-terminal         truncations thereof, or a sequence of at least about 80%         identity to any of them;     -   (iii) an N2 amino acid sequence of 0 to about 3 amino acids,         wherein each amino acid of the N2 amino acid sequence is among         the 12 most frequently occurring amino acids at the         corresponding position in N2 amino acid sequences of CDRH3 amino         acid sequences that are functionally expressed by human B cells;         and     -   (iv) a human CDRH3 H3-JH amino acid sequence, N-terminal         truncations thereof, or a sequence of at least about 80%         identity to any of them.

The following embodiments may be applied throughout the embodiments of the instant invention. In one aspect, one or more CDRH3 amino acid sequences further comprise an N-terminal tail residue. In still another aspect, the N-terminal tail residue is selected from the group consisting of G, D, and E.

In yet another aspect, the N1 amino acid sequence is selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof. In certain other aspects, the N1 amino acid sequence may be of about 0 to about 5 amino acids.

In yet another aspect, the N2 amino acid sequence is selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof. In certain other aspects, the N2 sequence may be of about 0 to about 5 amino acids.

In yet another aspect, the H3-JH amino acid sequence is selected from the group consisting of AEYFQH (SEQ ID NO: 17), EYFQH, YFQH, FQH, QH, H, YWYFDL (SEQ ID NO: 18), WYFDL, YFDL, FDL, DL, L, AFDV (SEQ ID NO: 19), FDV, DV, V, YFDY (SEQ ID NO: 20), FDY, DY, Y, NWFDS (SEQ ID NO: 21), WFDS, FDS, DS, S, YYYYYGMDV (SEQ ID NO: 22), YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, and MDV.

In other embodiments, the invention comprises a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at least one of the following i-i+1 pairs in the library is within the ranges specified below:

-   -   Tyr-Tyr in an amount from about 2.5% to about 6.5%;     -   Ser-Gly in an amount from about 2.5% to about 4.5%;     -   Ser-Ser in an amount from about 2% to about 4%;     -   Gly-Ser in an amount from about 1.5% to about 4%;     -   Tyr-Ser in an amount from about 0.75% to about 2%;     -   Tyr-Gly in an amount from about 0.75% to about 2%; and     -   Ser-Tyr in an amount from about 0.75% to about 2%.

In still other embodiments, the invention comprises a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at least one of the following i-i+2 pairs in the library is within the ranges specified below:

-   -   Tyr-Tyr in an amount from about 2.5% to about 4.5%;     -   Gly-Tyr in an amount from about 2.5% to about 5.5%;     -   Ser-Tyr in an amount from about 2% to about 4%;     -   Tyr-Ser in an amount from about 1.75% to about 3.75%;     -   Ser-Gly in an amount from about 2% to about 3.5%;     -   Ser-Ser in an amount from about 1.5% to about 3%;     -   Gly-Ser in an amount from about 1.5% to about 3%; and     -   Tyr-Gly in an amount from about 1% to about 2%.

In another embodiment, the invention comprises a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at least one of the following i-i+3 pairs in the library is within the ranges specified below:

-   -   Gly-Tyr in an amount from about 2.5% to about 6.5%;     -   Ser-Tyr in an amount from about 1% to about 5%;     -   Tyr-Ser in an amount from about 2% to about 4%;     -   Ser-Ser in an amount from about 1% to about 3%;     -   Gly-Ser in an amount from about 2% to about 5%; and     -   Tyr-Tyr in an amount from about 0.75% to about 2%.

In one aspect of the invention, at least 2, 3, 4, 5, 6, or 7 of the specified i-i+1 pairs in the library are within the specified ranges. In another aspect, the CDRH3 amino acid sequences are human. In yet another aspect, the polynucleotides encode at least about 10⁶ unique CDRH3 amino acid sequences.

In other aspects of the invention, the polynucleotides further encode one or more heavy chain chassis amino acid sequences that are N-terminal to the CDRH3 amino acid sequences, and the one or more heavy chain chassis sequences are selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 94 encoded by IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B, IGHV5-51, IGHV6-1, and IGHV7-4-1, or a sequence of at least about 80% identity to any of them.

In another aspect, the polynucleotides further encode one or more FRM4 amino acid sequences that are C-terminal to the CDRH3 amino acid sequences, wherein the one or more FRM4 amino acid sequences are selected from the group consisting of a FRM4 amino acid sequence encoded by IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6, or a sequence of at least about 80% identity to any of them. In still another aspect, the polynucleotides further encode one or more immunoglobulin heavy chain constant region amino acid sequences that are C-terminal to the FRM4 sequence.

In yet another aspect, the CDRH3 amino acid sequences are expressed as part of full-length heavy chains. In other aspects, the full-length heavy chains are selected from the group consisting of an IgG1, IgG2, IgG3, and IgG4, or combinations thereof. In one embodiment, the CDRH3 amino acid sequences are from about 2 to about 30, from about 8 to about 19, or from about 10 to about 18 amino acid residues in length. In other aspects, the synthetic polynucleotides of the library encode from about 10⁶ to about 10¹⁴, from about 10⁷ to about 10¹³, from about 10⁸ to about 10¹², from about 10⁹ to about 10¹², or from about 10¹⁰ to about 10¹² unique CDRH3 amino acid sequences.

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody VKCDR3 amino acid sequences comprising about 1 to about 10 of the amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95A, 96, and 97, in selected VKCDR3 amino acid sequences derived from a particular IGKV or IGKJ germline sequence.

In one aspect, the synthetic polynucleotides encode one or more of the amino acid sequences listed in Table 33 or a sequence at least about 80% identical to any of them.

In some embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of unique antibody VKCDR3 amino acid sequences that are of at least about 80% identity to an amino acid sequence represented by the following formula:

[VK_Chassis]-[L3-VK]-[X]-[JK*], wherein:

-   -   (i) VK_Chassis is an amino acid sequence selected from the group         consisting of about Kabat amino acid 1 to about Kabat amino acid         88 encoded by IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12,         IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37,         IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24,         IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29,         IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11,         IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41, or a         sequence of at least about 80% identity to any of them;     -   (ii) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene         segment; and     -   (iii) X is any amino acid residue; and     -   (iv) JK* is an amino acid sequence selected from the group         consisting of sequences encoded by IGJK1, IGJK2, IGJK3, IGJK4,         and IGJK5, wherein the first residue of each IGJK sequence is         not present.

In still other aspects, X may be selected from the group consisting of F, L, I, R, W, Y, and P.

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of VλCDR3 amino acid sequences that are of at least about 80% identity to an amino acid sequence represented by the following formula:

[Vλ_Chassis]-[L3-Vλ]-[Jλ], wherein:

-   -   (i) Vλ_Chassis is an amino acid sequence selected from the group         consisting of about Kabat amino acid 1 to about Kabat amino acid         88 encoded by IGλV1-36, IGλV1-40, IGλV1-44, IGλV1-47, IGλV1-51,         IGλV10-54, IGλV2-11, IGλV2-14, IGλV2-18, IGλV2-23, IGλV2-8,         IGλV3-1, IGλV3-10, IGλV3-12, IGλV3-16, IGλV3-19, IGλV3-21,         IGλV3-25, IGλV3-27, IGλV3-9, IGλV4-3, IGλV4-60, IGλV4-69,         IGλV5-39, IGλV5-45, IGλV6-57, IGλV7-43, IGλV7-46, IGλV8-61,         IGλV9-49, and IGλV10-54, or a sequence of at least about 80%         identity to any of them;     -   (ii) L3-Vλ is the portion of the VλCDR3 encoded by the IGλV         segment; and     -   (iii) Jλ is an amino acid sequence selected from the group         consisting of sequences encoded by IGλJ1-01, IGλJ2-01, IGλJ3-01,         IGλJ3-02, IGλJ6-01, IGλJ7-01, and IGλJ7-02, and wherein the         first residue of each IGJλ sequence may or may not be deleted.

In further aspects, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising:

-   -   (i) a CDRH3 amino acid sequence of claim 1; and     -   (ii) a VKCDR3 amino acid sequence comprising about 1 to about 10         of the amino acids found at Kabat positions 89, 90, 91, 92, 93,         94, 95, 95A, 96, and 97, in selected VKCDR3 sequences derived         from a particular IGKV or IGKJ germline sequence.

In still further aspects, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising:

-   -   (i) a CDRH3 amino acid sequence of claim 1; and     -   (ii) a VKCDR3 amino acid sequences of at least about 80%         identity to an amino acid sequence represented by the following         formula:

[VK_Chassis]-[L3-VK]-[X]-[JK*], wherein:

-   -   -   (a) VK_Chassis is an amino acid sequence selected from the             group consisting of about Kabat amino acid 1 to about Kabat             amino acid 88 encoded by IGKV1-05, IGKV1-06, IGKV1-08,             IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27,             IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17,             IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30,             IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11,             IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20,             IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41, or a sequence of             at least about 80% identity to any of them;         -   (b) L3-VK is the portion of the VKCDR3 encoded by the IGKV             gene segment; and         -   (c) X is any amino acid residue; and         -   (d) JK* is an amino acid sequence selected from the group             consisting of sequences encoded by IGJK1, IGJK2, IGJK3,             IGJK4, and IGJK5, wherein the first residue of each IGJK             sequence is not present.

In some aspects, the VKCDR3 amino acid sequence comprises one or more of the sequences listed in Table 33 or a sequence at least about 80% identical to any of them. In other aspects, the antibody proteins are expressed in a heterodimeric form. In yet another aspect, the human antibody proteins are expressed as antibody fragments. In still other aspects of the invention, the antibody fragments are selected from the group consisting of Fab, Fab′, F(ab′)₂, Fv fragments, diabodies, linear antibodies, and single-chain antibodies.

In certain embodiments, the invention comprises an antibody isolated from the polypeptide expression products of any library described herein.

In still other aspects, the polynucleotides further comprise a 5′ polynucleotide sequence and a 3′ polynucleotide sequence that facilitate homologous recombination.

In one embodiment, the polynucleotides further encode an alternative scaffold.

In another embodiment, the invention comprises a library of polypeptides encoded by any of the synthetic polynucleotide libraries described herein.

In yet another embodiment, the invention comprises a library of vectors comprising any of the polynucleotide libraries described herein. In certain other aspects, the invention comprises a population of cells comprising the vectors of the instant invention.

In one aspect, the doubling time of the population of cells is from about 1 to about 3 hours, from about 3 to about 8 hours, from about 8 to about 16 hours, from about 16 to about 20 hours, or from 20 to about 30 hours. In yet another aspect, the cells are yeast cells. In still another aspect, the yeast is Saccharomyces cerevisiae.

In other embodiments, the invention comprises a library that has a theoretical total diversity of N unique CDRH3 sequences, wherein N is about 10⁶ to about 10¹⁵; and wherein the physical realization of the theoretical total CDRH3 diversity has a size of at least about 3N, thereby providing a probability of at least about 95% that any individual CDRH3 sequence contained within the theoretical total diversity of the library is present in the actual library.

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody VλCDR3 amino acid sequences comprising about 1 to about 10 of the amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95A, 95B, 95C, 96, and 97, in selected VλCDR3 sequences encoded by a single germline sequence.

In some embodiments, the invention relates to a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the library has a theoretical total diversity of about 10⁶ to about 10¹⁵ unique CDRH3 sequences.

In still other embodiments, the invention relates to a method of preparing a library of synthetic polynucleotides encoding a plurality of antibody VK amino acid sequences, the method comprising:

(i) providing polynucleotide sequences encoding:

-   -   (a) one or more VK_Chassis amino acid sequences selected from         the group consisting of about Kabat amino acid 1 to about Kabat         amino acid 88 encoded by IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09,         IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33,         IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8,         IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26,         IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07,         IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41,         or a sequence at least about 80% identical to any of them;     -   (b) one or more L3-VK amino acid sequences, wherein L3-VK the         portion of the VKCDR3 amino acid sequence encoded by the IGKV         gene segment;     -   (c) one or more X residues, wherein X is any amino acid residue;         and     -   (d) one or more JK* amino acid sequences, wherein JK* is an         amino acid sequence selected from the group consisting amino         acid sequences encoded by IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5,         wherein the first amino acid residue of each sequence is not         present; and

(ii) assembling the polynucleotide sequences to produce a library of synthetic polynucleotides encoding a plurality of human VK sequences represented by the following formula:

[VK_Chassis]-[L3-VK]-[X]-[JK*].

In some embodiments, the invention relates to a method of preparing a library of synthetic polynucleotides encoding a plurality of antibody light chain CDR3 sequences, the method comprising:

(i) determining the percent occurrence of each amino acid residue at each position in selected light chain CDR3 amino acid sequences derived from a single germline polynucleotide sequence;

(ii) designing synthetic polynucleotides encoding a plurality of human antibody light chain CDR3 amino acid sequences, wherein the percent occurrence of any amino acid at any position within the designed light chain CDR3 amino acid sequences is within at least about 30% of the percent occurrence in the selected light chain CDR3 amino acid sequences derived from a single germline polynucleotide sequence, as determined in (i); and

(iii) synthesizing one or more polynucleotides that were designed in (ii).

In other embodiments, the invention relates to a method of preparing a library of synthetic polynucleotides encoding a plurality of antibody VP amino acid sequences, the method comprising:

(i) providing polynucleotide sequences encoding:

-   -   (a) one or more Vλ_Chassis amino acid sequences selected from         the group consisting of about Kabat residue 1 to about Kabat         residue 88 encoded by IGλV1-36, IGλV1-40, IGλV1-44, IGλV1-47,         IGλV1-51, IGλV10-54, IGλV2-11, IGλV2-14, IGλV2-18, IGλV2-23,         IGλV2-8, IGλV3-1, IGλV3-10, IGλV3-12, IGλV3-16, IGλV3-19,         IGλV3-21, IGλV3-25, IGλV3-27, IGλV3-9, IGλV4-3, IGλV4-60,         IGλV4-69, IGλV5-39, IGλV5-45, IGλV6-57, IGλV7-43, IGλV7-46,         IGλV8-61, IGλV9-49, and IGλV10-54, or a sequence at least about         80% identical to any of them;     -   (b) one or more L3-VX sequences, wherein L3-VX is the portion of         the VλCDR3 amino acid sequence encoded by the IGλV gene segment;     -   (c) one or more Jλ sequences, wherein Jλ is an amino acid         sequence selected from the group consisting of amino acid         sequences encoded by IGλJ1-01, IGλJ2-01, IGλJ3-01, IGλJ3-02,         IGλJ6-01, IGλJ7-01, and IGλJ7-02 wherein the first amino acid         residue of each sequence may or may not be present; and

(ii) assembling the polynucleotide sequences to produce a library of synthetic polynucleotides encoding a plurality of human VX amino acid sequences represented by the following formula:

[Vλ_Chassis]-[L3-Vλ]-[Jλ].

In certain embodiments, the amino acid sequences encoded by the polynucleotides of the libraries of the invention are human.

The present invention is also directed to methods of preparing a synthetic polynucleotide library comprising providing and assembling the polynucleotide sequences of the instant invention.

In another aspect, the invention comprises a method of preparing the library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, the method comprising:

-   -   (i) providing polynucleotide sequences encoding:         -   (a) one or more N1 amino acid sequences of about 0 to about             3 amino acids, wherein each amino acid of the N1 amino acid             sequence is among the 12 most frequently occurring amino             acids at the corresponding position in N1 sequences of CDRH3             amino acid sequences that are functionally expressed by             human B cells;         -   (b) one or more human CDRH3 DH amino acid sequences, N- and             C-terminal truncations thereof, or a sequence of at least             about 80% identity to any of them;         -   (c) one or more N2 amino acid sequences of about 0 to about             3 amino acids, wherein each amino acid of the N1 amino acid             sequence is among the 12 most frequently occurring amino             acids at the corresponding position in N2 amino acid             sequences of CDRH3 amino acid sequences that are             functionally expressed by human B cells; and         -   (d) one or more human CDRH3 H3-JH amino acid sequences,             N-terminal truncations thereof, or a sequence of at least             about 80% identity to any of them; and     -   (ii) assembling the polynucleotide sequences to produce a         library of synthetic polynucleotides encoding a plurality of         human antibody CDRH3 amino acid sequences represented by the         following formula:

[N1]-[DH]-[N2]-[H3-JH].

In one aspect, one or more of the polynucleotide sequences are synthesized via split-pool synthesis.

In another aspect, the method of the invention further comprises the step of recombining the assembled synthetic polynucleotides with a vector comprising a heavy chain chassis and a heavy chain constant region, to form a full-length heavy chain.

In another aspect, the method of the invention further comprises the step of providing a 5′ polynucleotide sequence and a 3′ polynucleotide sequence that facilitate homologous recombination. In still another aspect, the method of the invention further comprises the step of recombining the assembled synthetic polynucleotides with a vector comprising a heavy chain chassis and a heavy chain constant region, to form a full-length heavy chain.

In some embodiments, the step of recombining is performed in yeast. In certain embodiments, the yeast is S. cerevisiae.

In certain other embodiments, the invention comprises a method of isolating one or more host cells expressing one or more antibodies, the method comprising:

-   -   (i) expressing the human antibodies of any one of claims 40 and         46 in one or more host cells;     -   (ii) contacting the host cells with one or more antigens; and     -   (iii) isolating one or more host cells having antibodies that         bind to the one or more antigens.

In another aspect, the method of the invention further comprises the step of isolating one or more antibodies from the one or more host cells that present the antibodies which recognize the one or more antigens. In yet another aspect, the method of the invention further comprises the step of isolating one or more polynucleotide sequences encoding one or more antibodies from the one or more host cells that present the antibodies which recognize the one or more antigens.

In certain other embodiments, the invention comprises a kit comprising the library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, or any of the other sequences disclosed herein.

In still other aspects, the CDRH3 amino acid sequences encoded by the libraries of synthetic polynucleotides described herein, or any of the other sequences disclosed herein, are in computer readable form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of recombination between a fragment (e.g., CDR3) and a vector (e.g., comprising a chassis and constant region) for the construction of a library.

FIG. 2 depicts the length distribution of the N1 and N2 regions of rearranged human antibody sequences compiled from Jackson et al. (J. Immunol. Methods, 2007, 324: 26, incorporated by reference in its entirety).

FIG. 3 depicts the length distribution of the CDRL3 regions of rearranged human kappa light chain sequences compiled from the NCBI database (Appendix A).

FIG. 4 depicts the length distribution of the CDRL3 regions of rearranged human lambda light chain sequences compiled from the NCBI database (Appendix B).

FIG. 5 depicts a schematic representation of the 424 cloning vectors used in the synthesis of the CDRH3 regions before and after ligation of the [DH]-[N2]-[JH] segment (DTAVYYCAR: SEQ ID NO: 579; DTAVYYCAK: SEQ ID NO: 578; SSASTK: SEQ ID NO: 580).

FIG. 6 depicts a schematic structure of a heavy chain vector, prior to recombination with a CDRH3.

FIG. 7 depicts a schematic diagram of a CDRH3 integrated into a heavy chain vector and the polynucleotide and polypeptide sequences of CDRH3_(SEQ ID NO: δ 1).

FIG. 8 depicts a schematic structure of a kappa light chain vector, prior to recombination with a CDRL3.

FIG. 9 depicts a schematic diagram of a CDRL3 integrated into a light chain vector and the polynucleotide and polypeptide sequences of CDRL3 (SEQ ID NO: 582).

FIG. 10 depicts the length distribution of the CDRH3 domain (Kabat positions 95-102) from 96 colonies obtained by transformation with 10 of the 424 vectors synthesized as described in Example 10 (observed), as compared to the expected (i.e., designed) distribution.

FIG. 11 depicts the length distribution of the DH segment from 96 colonies obtained by transformation with 10 of the 424 vectors synthesized as described in Example 10 (observed), as compared to the expected (i.e., designed) distribution.

FIG. 12 depicts the length distribution of the N2 segment from 96 colonies obtained by transformation with 10 of the 424 vectors synthesized as described in Example 10 (observed), as compared to the expected (i.e., designed) distribution.

FIG. 13 depicts the length distribution of the H3-JH segment from 96 colonies obtained by transformation with 10 of the 424 vectors synthesized as described in Example 10 (observed), as compared to the expected (i.e., designed) distribution.

FIG. 14 depicts the length distribution of the CDRH3 domains from 291 sequences prepared from yeast cells transformed according to the method outlined in Example 10.4, namely the co-transformation of vectors containing heavy chain chassis and constant regions with a CDRH3 insert (observed), as compared to the expected (i.e., designed) distribution.

FIG. 15 depicts the length distribution of the [Tail]-[N1] region from the 291 sequences prepared from yeast cells transformed according to the protocol outlined in Example 10.4 (observed), as compared to the expected (i.e., designed) distribution.

FIG. 16 depicts the length distribution of the DH region from the 291 sequences prepared from yeast cells transformed according to the protocol outlined in Example 10.4 (observed), as compared to the theoretical (i.e., designed) distribution.

FIG. 17 depicts the length distribution of the N2 region from the 291 sequences prepared from yeast cells transformed according to the protocol outlined in Example 10.4 (observed), as compared to the theoretical (i.e., designed) distribution.

FIG. 18 depicts the length distribution of the H3-JH region from the 291 sequences prepared from yeast cells transformed according to the protocol outlined in Example 10.4 (observed), as compared to the theoretical (i.e., designed) distribution.

FIG. 19 depicts the familial origin of the JH segments identified in the 291 sequences (observed), as compared to the theoretical (i.e., designed) familial origin.

FIG. 20 depicts the representation of each of the 16 chassis of the library (observed), as compared to the theoretical (i.e., designed) chassis representation. VH3-23 is represented twice; once ending in CAR and once ending in CAK. These representations were combined, as were the ten variants of VH3-33 with one variant of VH3-30.

FIG. 21 depicts a comparison of the CDRL3 length from 86 sequences selected from the VKCDR3 library of Example 6.2 (observed) to human sequences (human) and the designed sequences (designed).

FIG. 22 depicts the representation of the light chain chassis amongst the 86 sequences selected from the library (observed), as compared to the theoretical (i.e., designed) chassis representation.

FIG. 23 depicts the frequency of occurrence of different CDRH3 lengths in an exemplary library of the invention, versus the preimmune repertoire of Lee et al. (Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety).

FIG. 24 depicts binding curves for 6 antibodies selected from a library of the invention.

FIG. 25 depicts binding data for 10 antibodies selected from a library of the invention binding to hen egg white lysozyme.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to, at least, synthetic polynucleotide libraries, methods of producing and using the libraries of the invention, kits and computer readable forms including the libraries of the invention. The libraries taught in this application are described, at least in part, in terms of the components from which they are assembled.

In certain embodiments, the instant invention provides antibody libraries specifically designed based on the composition and CDR length distribution in the naturally occurring human antibody repertoire. It is estimated that, even in the absence of antigenic stimulation, a human makes at least about 10⁷ different antibody molecules. The antigen-binding sites of many antibodies can cross-react with a variety of related but different epitopes. In addition the human antibody repertoire is large enough to ensure that there is an antigen-binding site to fit almost any potential epitope, albeit with low affinity.

The mammalian immune system has evolved unique genetic mechanisms that enable it to generate an almost unlimited number of different light and heavy chains in a remarkably economical way, by combinatorially joining chromosomally separated gene segments prior to transcription. Each type of immunoglobulin (Ig) chain (i.e., κ light, λ light, and heavy) is synthesized by combinatorial assembly of DNA sequences selected from two or more families of gene segments, to produce a single polypeptide chain. Specifically, the heavy chains and light chains each consist of a variable region and a constant (C) region. The variable regions of the heavy chains are encoded by DNA sequences assembled from three families of gene segments: variable (IGHV), joining (IGHJ) and diversity (IGHD). The variable regions of light chains are encoded by DNA sequences assembled from two families of gene segments for each of the kappa and lambda light chains: variable (IGLV) and joining (IGLJ). Each variable region (heavy and light) is also recombined with a constant region, to produce a full-length immunoglobulin chain.

While combinatorial assembly of the V, D and J gene segments make a substantial contribution to antibody variable region diversity, further diversity is introduced in vivo, at the pre-B cell stage, via imprecise joining of these gene segments and the introduction of non-templated nucleotides at the junctions between the gene segments.

After a B cell recognizes an antigen, it is induced to proliferate. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is far greater than the normal rate of genomic mutation. The mutations that occur are primarily localized to the Ig variable regions and comprise substitutions, insertions and deletions. This somatic hypermutation enables the production of B cells that express antibodies possessing enhanced affinity toward an antigen. Such antigen-driven somatic hypermutation fine-tunes antibody responses to a given antigen.

Significant efforts have been made to create antibody libraries with extensive diversity, and to mimic the natural process of affinity maturation of antibodies against various antigens, especially antigens associated with diseases such as autoimmune diseases, cancer, and infectious disease. Antibody libraries comprising candidate binding molecules that can be readily screened against targets are desirable. However, the full promise of an antibody library, which is representative of the preimmune human antibody repertoire, has remained elusive. In addition to the shortcomings enumerated above, and throughout the application, synthetic libraries that are known in the art often suffer from noise (i.e., very large libraries increase the presence of many sequences which do not express well, and/or which misfold), while entirely human libraries that are known in the art may be biased against certain antigen classes (e.g. self-antigens). Moreover, the limitations of synthesis and physical realization techniques restrict the functional diversity of antibody libraries of the art. The present invention provides, for the first time, a fully synthetic antibody library that is representative of the human preimmune antibody repertoire (e.g., in composition and length), and that can be readily screened (i.e., it is physically realizable and, in some cases can be oversampled) using, for example, high throughput methods, to obtain, for example, new therapeutics and/or diagnostics

In particular, the synthetic antibody libraries of the instant invention have the potential to recognize any antigen, including self-antigens of human origin. The ability to recognize self-antigens is usually lost in an expressed human library, because self-reactive antibodies are removed by the donor's immune system via negative selection. Another feature of the invention is that screening the antibody library using positive clone selection, for example, by FACS (florescence activated cell sorter) bypasses the standard and tedious methodology of generating a hybridoma library and supernatant screening. Still further, the libraries, or sub-libraries thereof, can be screened multiple times, to discover additional antibodies against other desired targets.

Before further description of the invention, certain terms are defined.

1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art relevant to the invention. The definitions below supplement those in the art and are directed to the embodiments described in the current application.

The term “antibody” is used herein in the broadest sense and specifically encompasses at least monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g. bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, and antibody fragments. An antibody is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.

“Antibody fragments” comprise a portion of an intact antibody, for example, one or more portions of the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments, diabodies, linear antibodies, single-chain antibodies, and multi-specific antibodies formed from intact antibodies and antibody fragments.

An “intact antibody” is one comprising full-length heavy- and light-chains and an Fc region. An intact antibody is also referred to as a “full-length, heterodimeric” antibody or immunoglobulin.

The term “variable” refers to the portions of the immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (i.e., the “variable domain(s)”). Variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable regions. These sub-domains are called “hypervariable” regions or “complementarity determining regions” (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called the “framework” regions (FRM). The variable domains of naturally occurring heavy and light chains each comprise four FRM regions, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRM and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site (see Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991, incorporated by reference in its entirety). The constant domains are not directly involved in antigen binding, but exhibit various effector functions, such as, for example, antibody-dependent, cell-mediated cytotoxicity and complement activation.

The “chassis” of the invention represent a portion of the antibody heavy chain variable (IGHV) or light chain variable (IGLV) domains that are not part of CDRH3 or CDRL3, respectively. The chassis of the invention is defined as the portion of the variable region of an antibody beginning with the first amino acid of FRM1 and ending with the last amino acid of FRM3. In the case of the heavy chain, the chassis includes the amino acids including from about Kabat position 1 to about Kabat position 94. In the case of the light chains (kappa and lambda), the chassis are defined as including from about Kabat position 1 to about Kabat position 88. The chassis of the invention may contain certain modifications relative to the corresponding germline variable domain sequences presented herein or available in public databases. These modifications may be engineered (e.g., to remove N-linked glycosylation sites) or naturally occurring (e.g. to account for allelic variation). For example, it is known in the art that the immunoglobulin gene repertoire is polymorphic (Wang et al., Immunol. Cell. Biol., 2008, 86: 111; Collins et al., Immunogenetics, 2008, DOI 10.1007/s00251-008-0325-z, published online, each incorporated by reference in its entirety); chassis, CDRs (e.g., CDRH3) and constant regions representative of these allelic variants are also encompassed by the invention. In some embodiments, the allelic variant(s) used in a particular embodiment of the invention may be selected based on the allelic variation present in different patient populations, for example, to identify antibodies that are non-immunogenic in these patient populations. In certain embodiments, the immunogenicity of an antibody of the invention may depend on allelic variation in the major histocompatibility complex (MHC) genes of a patient population. Such allelic variation may also be considered in the design of libraries of the invention. In certain embodiments of the invention, the chassis and constant regions are contained on a vector, and a CDR3 region is introduced between them via homologous recombination.

In some embodiments, one, two or three nucleotides may follow the heavy chain chassis, forming either a partial (if one or two) or a complete (if three) codon. When a full codon is present, these nucleotides encode an amino acid residue that is referred to as the “tail,” and occupies position 95.

The “CDRH3 numbering system” used herein defines the first amino acid of CDRH3 as being at Kabat position 95 (the “tail,” when present) and the last amino acid of CDRH3 as position 102. The amino acids following the “tail” are called “N1” and, when present, are assigned numbers 96, 96A, 96B, etc. The N1 segment is followed by the “DH” segment, which is assigned numbers 97, 97A, 97B, 97C, etc. The DH segment is followed by the “N2” segment, which, when present, is numbered 98, 98A, 98B, etc. Finally, the most C-terminal amino acid residue of the set of the “H3-JH” segment is designated as number 102. The residue directly before (N-terminal) it, when present, is 101, and the one before (if present) is 100. For reasons of convenience, and which will become apparent elsewhere, the rest of the H3-JH amino acids are numbered in reverse order, beginning with 99 for the amino acid just N-terminal to 100, 99A for the residue N-terminal to 99, and so forth for 99B, 99C, etc. Examples of certain CDRH3 sequence residue numbers may therefore include the following:

As used herein, the term “diversity” refers to a variety or a noticeable heterogeneity. The term “sequence diversity” refers to a variety of sequences which are collectively representative of several possibilities of sequences, for example, those found in natural human antibodies. For example, heavy chain CDR3 (CDRH3) sequence diversity may refer to a variety of possibilities of combining the known human DH and H3-JH segments, including the N1 and N2 regions, to form heavy chain CDR3 sequences. The light chain CDR3 (CDRL3) sequence diversity may refer to a variety of possibilities of combining the naturally occurring light chain variable region contributing to CDRL3 (i.e., L3-VL) and joining (i.e., L3-JL) segments, to form light chain CDR3 sequences. As used herein, H3-JH refers to the portion of the IGHJ gene contributing to CDRH3. As used herein, L3-VL and L3-JL refer to the portions of the IGLV and IGLJ genes (kappa or lambda) contributing to CDRL3, respectively.

As used herein, the term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used herein, the term “host cell” is intended to refer to a cell into which a polynucleotide of the invention. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The term “length diversity” refers to a variety in the length of a particular nucleotide or amino acid sequence. For example, in naturally occurring human antibodies, the heavy chain CDR3 sequence varies in length, for example, from about 3 amino acids to over about 35 amino acids, and the light chain CDR3 sequence varies in length, for example, from about 5 to about 16 amino acids. Prior to the instant invention, it was known in the art that it is possible to design antibody libraries containing sequence diversity or length diversity (see, e.g., Hoet et al., Nat. Biotechnol., 2005, 23: 344; Kretzschmar and von Ruden, Curr. Opin. Biotechnol., 2002 13: 598; and Rauchenberger et al., J. Biol. Chem., 2003 278: 38194, each of which is incorporated by reference in its entirety); however, the instant invention is directed to, at least, the design of synthetic antibody libraries containing the sequence diversity and length diversity of naturally occurring human sequences. In some cases, synthetic libraries containing sequence and length diversity have been synthesized, however these libraries contain too much theoretical diversity to synthesize the entire designed repertoire and/or too many theoretical members to physically realize or oversample the entire library.

As used herein, a sequence designed with “directed diversity” has been specifically designed to contain both sequence diversity and length diversity. Directed diversity is not stochastic.

As used herein, “stochastic” describes a process of generating a randomly determined sequence of amino acids, which is considered as a sample of one element from a probability distribution.

The term “library of polynucleotides” refers to two or more polynucleotides having a diversity as described herein, specifically designed according to the methods of the invention. The term “library of polypeptides” refers to two or more polypeptides having a diversity as described herein, specifically designed according to the methods of the invention. The term “library of synthetic polynucleotides” refers to a polynucleotide library that includes synthetic polynucleotides. The term “library of vectors” refers herein to a library of at least two different vectors. As used herein, the term “human antibody libraries,” at least includes, a polynucleotide or polypeptide library which has been designed to represent the sequence diversity and length diversity of naturally occurring human antibodies.

As described throughout the specification, the term “library” is used herein in its broadest sense, and also may include the sub-libraries that may or may not be combined to produce libraries of the invention.

As used herein, the term “synthetic polynucleotide” refers to a molecule formed through a chemical process, as opposed to molecules of natural origin, or molecules derived via template-based amplification of molecules of natural origin (e.g., immunoglobulin chains cloned from populations of B cells via PCR amplification are not “synthetic” used herein). In some instances, for example, when referring to libraries of the invention that comprise multiple components (e.g., N1, DH, N2, and/or H3-JH), the invention encompasses libraries in which at least one of the aforementioned components is synthetic. By way of illustration, a library in which certain components are synthetic, while other components are of natural origin or derived via template-based amplification of molecules of natural origin, would be encompassed by the invention.

The term “split-pool synthesis” refers to a procedure in which the products of a plurality of first reactions are combined (pooled) and then separated (split) before participating in a plurality of second reactions. Example 9, describes the synthesis of 278 DH segments (products), each in a separate reaction. After synthesis, these 278 segments are combined (pooled) and then distributed (split) amongst 141 columns for the synthesis of the N2 segments. This enables the pairing of each of the 278 DH segments with each of the 141 N2 segments. As described elsewhere in the specification, these numbers are non-limiting.

“Preimmune” antibody libraries have similar sequence diversities and length diversities to naturally occurring human antibody sequences before these sequences have undergone negative selection or somatic hypermutation. For example, the set of sequences described in Lee et al. (Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety) is believed to represent sequences from the preimmune repertoire. In certain embodiments of the invention, the sequences of the invention will be similar to these sequences (e.g., in terms of composition and length). In certain embodiments of the invention, such antibody libraries are designed to be small enough to chemically synthesize and physically realize, but large enough to encode antibodies with the potential to recognize any antigen. In one embodiment of the invention, an antibody library comprises about 10⁷ to about 10²⁰ different antibodies and/or polynucleotide sequences encoding the antibodies of the library. In some embodiments, the libraries of the instant invention are designed to include 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, or 10²⁰ different antibodies and/or polynucleotide sequences encoding the antibodies. In certain embodiments, the libraries of the invention may comprise or encode about 10³ to about 10⁵, about 10⁵ to about 10⁷, about 10⁷ to about 10⁹, about 10⁹ to about 10¹¹, about 10¹¹ to about 10¹³, about 10¹³ to about 10¹⁵, about 10¹⁵ to about 10¹⁷, or about 10¹⁷ to about 10²⁰ different antibodies. In certain embodiments of the invention, the diversity of the libraries may be characterized as being greater than or less than one or more of the diversities enumerated above, for example greater than about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, or 10²⁰ or less than about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, or 10²⁰. In certain other embodiments of the invention, the probability of an antibody of interest being present in a physical realization of a library with a size as enumerated above is at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% (see Library Sampling, in the Detailed Description, for more information on the probability of a particular sequence being present in a physical realization of a library). The antibody libraries of the invention may also include antibodies directed to, for example, self (i.e., human) antigens. The antibodies of the present invention may not be present in expressed human libraries for reasons including because self-reactive antibodies are removed by the donor's immune system via negative selection. However, novel heavy/light chain pairings may in some cases create self-reactive antibody specificity (Griffiths et al. U.S. Pat. No. 5,885,793, incorporated by reference in its entirety). In certain embodiments of the invention, the number of unique heavy chains in a library may be about 10, 50, 10², 150, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more. In certain embodiments of the invention, the number of unique light chains in a library may be about 5, 10, 25, 50, 10², 150, 500, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more.

As used herein, the term “human antibody CDRH3 libraries,” at least includes, a polynucleotide or polypeptide library which has been designed to represent the sequence diversity and length diversity of naturally occurring human antibodies. “Preimmune” CDRH3 libraries have similar sequence diversities and length diversities to naturally occurring human antibody CDRH3 sequences before these sequences undergo negative selection and somatic hypermutation. Known human CDRH3 sequences are represented in various data sets, including Jackson et al., J. Immunol. Methods, 2007, 324: 26; Martin, Proteins, 1996, 25: 130; and Lee et al., Immunogenetics, 2006, 57: 917, each of which is incorporated by reference in its entirety. In certain embodiments of the invention, such CDRH3 libraries are designed to be small enough to chemically synthesize and physically realize, but large enough to encode CDRH3s with the potential to recognize any antigen. In one embodiment of the invention, an antibody library includes about 10⁶ to about 10¹⁵ different CDRH3 sequences and/or polynucleotide sequences encoding said CDRH3 sequences. In some embodiments, the libraries of the instant invention are designed to about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, or 10¹⁶, different CDRH3 sequences and/or polynucleotide sequences encoding said CDRH3 sequences. In some embodiments, the libraries of the invention may include or encode about 10³ to about 10⁶, about 10⁶ to about 10⁸, about 10⁸ to about 10¹⁰, about 10¹⁰ to about 10¹², about 10¹² to about 10¹⁴, or about 10¹⁴ to about 10¹⁶ different CDRH3 sequences. In certain embodiments of the invention, the diversity of the libraries may be characterized as being greater than or less than one or more of the diversities enumerated above, for example greater than about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, or 10¹⁶ or less than about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, or 10¹⁶. In certain embodiments of the invention, the probability of a CDRH3 of interest being present in a physical realization of a library with a size as enumerated above is at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% (see Library Sampling, in the Detailed Description, for more information on the probability of a particular sequence being present in a physical realization of a library). The preimmune CDRH3 libraries of the invention may also include CDRH3s directed to, for example, self (i.e., human) antigens. Such CDRH3s may not be present in expressed human libraries, because self-reactive CDRH3s are removed by the donor's immune system via negative selection.

Libraries of the invention containing “VKCDR3” sequences and “VλCDR3” sequences refer to the kappa and lambda sub-sets of the CDRL3 sequences, respectively. These libraries may be designed with directed diversity, to collectively represent the length and sequence diversity of the human antibody CDRL3 repertoire. “Preimmune” versions of these libraries have similar sequence diversities and length diversities to naturally occurring human antibody CDRL3 sequences before these sequences undergo negative selection. Known human CDRL3 sequences are represented in various data sets, including the NCBI database (see Appendix A and Appendix B for light chain sequence data sets) and Martin, Proteins, 1996, 25: 130 incorporated by reference in its entirety. In certain embodiments of the invention, such CDRL3 libraries are designed to be small enough to chemically synthesize and physically realize, but large enough to encode CDRL3s with the potential to recognize any antigen.

In one embodiment of the invention, an antibody library comprises about 10⁵ different CDRL3 sequences and/or polynucleotide sequences encoding said CDRL3 sequences. In some embodiments, the libraries of the instant invention are designed to comprise about 10¹, 10², 10³, 10⁴, 10⁶, 10⁷, or 10⁸ different CDRL3 sequences and/or polynucleotide sequences encoding said CDRL3 sequences. In some embodiments, the libraries of the invention may comprise or encode about 10¹ to about 10³, about 10³ to about 10⁵, or about 10⁵ to about 10⁸ different CDRL3 sequences. In certain embodiments of the invention, the diversity of the libraries may be characterized as being greater than or less than one or more of the diversities enumerated above, for example greater than about 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ or less than about 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸. In certain embodiments of the invention, the probability of a CDRL3 of interest being present in a physical realization of a library with a size as enumerated above is at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% (see Library Sampling, in the Detailed Description, for more information on the probability of a particular sequence being present in a physical realization of a library). The preimmune CDRL3 libraries of the invention may also include CDRL3s directed to, for example, self (i.e., human) antigens. Such CDRL3s may not be present in expressed human libraries, because self-reactive CDRL3s are removed by the donor's immune system via negative selection.

As used herein, the term “known heavy chain CDR3 sequences” refers to heavy chain CDR3 sequences in the public domain that have been cloned from populations of human B cells. Examples of such sequences are those published or derived from public data sets, including, for example, Zemlin et al., JMB, 2003, 334: 733; Lee et al., Immunogenetics, 2006, 57: 917; and Jackson et al. J. Immunol. Methods, 2007, 324: 26, each of which are incorporated by reference in their entirety.

As used herein, the term “known light chain CDR3 sequences” refers to light chain CDR3 sequences (e.g., kappa or lambda) in the public domain that have been cloned from populations of human B cells. Examples of such sequences are those published or derived from public data sets, including, for example, the NCBI database (see Appendices A and B filed herewith).

As used herein the term “antibody binding regions” refers to one or more portions of an immunoglobulin or antibody variable region capable of binding an antigen(s). Typically, the antibody binding region is, for example, an antibody light chain (or variable region or one or more CDRs thereof), an antibody heavy chain (or variable region or one or more CDRs thereof), a heavy chain Fd region, a combined antibody light and heavy chain (or variable regions thereof) such as a Fab, F(ab′)₂, single domain, or single chain antibodies (scFv), or any region of a full length antibody that recognizes an antigen, for example, an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

The term “framework region” refers to the art-recognized portions of an antibody variable region that exist between the more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 through 4 (FRM1, FRM2, FRM3, and FRM4) and provide a scaffold for the presentation of the six CDRs (three from the heavy chain and three from the light chain) in three dimensional space, to form an antigen-binding surface.

The term “canonical structure” refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothia et al., Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol., 1996, 263: 800, each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework (i.e., outside of the loop). Assignment to a particular canonical class can therefore be made based on the presence of these key amino acid residues. The term “canonical structure” may also include considerations as to the linear sequence of the antibody, for example, as catalogued by Kabat (Kabat et al., in “Sequences of Proteins of Immunological Interest,” 5^(th) Edition, U.S. Department of Heath and Human Services, 1992). The Kabat numbering scheme is a widely adopted standard for numbering the amino acid residues of an antibody variable domain in a consistent manner. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, those differences not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al. and/or revealed by other techniques, for example, crystallography and two or three-dimensional computational modeling. Accordingly, a given antibody sequence may be placed into a canonical class which allows for, among other things, identifying appropriate chassis sequences (e.g., based on a desire to include a variety of canonical structures in a library). Kabat numbering of antibody amino acid sequences and structural considerations as described by Chothia et al., and their implications for construing canonical aspects of antibody structure, are described in the literature.

The terms “CDR”, and its plural “CDRs”, refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDRL1, CDRL2 and CDRL3) and three make up the binding character of a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Kabat, Chothia, contact or any other boundary definitions, including the numbering system described herein. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called “hypervariable regions” within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent framework region. See for example Kabat, Chothia, and/or MacCallum et al., (Kabat et al., in “Sequences of Proteins of Immunological Interest,” 5^(th) Edition, U.S. Department of Health and Human Services, 1992; Chothia et al., J. Mol. Biol., 1987, 196: 901; and MacCallum et al., J. Mol. Biol., 1996, 262: 732, each of which is incorporated by reference in its entirety).

The term “amino acid” or “amino acid residue” typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

The term “polynucleotide(s)” refers to nucleic acids such as DNA molecules and RNA molecules and analogs thereof (e.g., DNA or RNA generated using nucleotide analogs or using nucleic acid chemistry). As desired, the polynucleotides may be made synthetically, e.g. using art-recognized nucleic acid chemistry or enzymatically using, e.g. a polymerase, and, if desired, be modified. Typical modifications include methylation, biotinylation, and other art-known modifications. In addition, the nucleic acid molecule can be single-stranded or double-stranded and, where desired, linked to a detectable moiety.

The terms “theoretical diversity”, “theoretical total diversity”, or “theoretical repertoire” refer to the maximum number of variants in a library design. For example, given an amino acid sequence of three residues, where residues one and three may each be any one of five amino acid types and residue two may be any one of 20 amino acid types, the theoretical diversity is 5×20×5=500 possible sequences. Similarly if sequence X is constructed by combination of 4 amino acid segments, where segment 1 has 100 possible sequences, segment 2 has 75 possible sequences, segment 3 has 250 possible sequences, and segment 4 has 30 possible sequences, the theoretical total diversity of fragment X would be 100×75×200×30, or 5.6×10⁵ possible sequences.

The term “physical realization” refers to a portion of the theoretical diversity that can actually be physically sampled, for example, by any display methodology. Exemplary display methodology include: phage display, ribosomal display, and yeast display. For synthetic sequences, the size of the physical realization of a library depends on (1) the fraction of the theoretical diversity that can actually be synthesized, and (2) the limitations of the particular screening method. Exemplary limitations of screening methods include the number of variants that can be screened in a particular assay (e.g. ribosome display, phage display, yeast display) and the transformation efficiency of a host cell (e.g., yeast, mammalian cells, bacteria) which is used in a screening assay. For the purposes of illustration, given a library with a theoretical diversity of 10¹² members, an exemplary physical realization of the library (e.g., in yeast, bacterial cells, ribosome display, etc.; details provided below) that can maximally include 10¹¹ members will, therefore, sample about 10% of the theoretical diversity of the library. However, if less than 10¹¹ members of the library with a theoretical diversity of 10¹² are synthesized, and the physical realization of the library can maximally include 10¹¹ members, less than 10% of the theoretical diversity of the library is sampled in the physical realization of the library. Similarly, a physical realization of the library that can maximally include more than 10¹² members would “oversample” the theoretical diversity, meaning that each member may be present more than once (assuming that the entire 10¹² theoretical diversity is synthesized).

The term “all possible reading frames” encompasses at least the three forward reading frames and, in some embodiments, the three reverse reading frames.

The term “antibody of interest” refers to any antibody that has a property of interest that is isolated from a library of the invention. The property of interest may include, but is not limited to, binding to a particular antigen or epitope, blocking a binding interaction between two molecules, or eliciting a certain biological effect.

The term “functionally expressed” refers to those immunoglobulin genes that are expressed by human B cells and that do not contain premature stop codons.

The term “full-length heavy chain” refers to an immunoglobulin heavy chain that contains each of the canonical structural domains of an immunoglobulin heavy chain, including the four framework regions, the three CDRs, and the constant region. The term “full-length light chain” refers to an immunoglobulin light chain that contains each of the canonical structural domains of an immunoglobulin light chain, including the four framework regions, the three CDRs, and the constant region.

The term “unique,” as used herein, refers to a sequence that is different (e.g. has a different chemical structure) from every other sequence within the designed theoretical diversity. It should be understood that there are likely to be more than one copy of many unique sequences from the theoretical diversity in a particular physical realization. For example, a library comprising three unique sequences may comprise nine total members if each sequence occurs three times in the library. However, in certain embodiments, each unique sequence may occur only once.

The term “heterologous moiety” is used herein to indicate the addition of a composition to an antibody wherein the composition is not normally part of the antibody. Exemplary heterologous moieties include drugs, toxins, imaging agents, and any other compositions which might provide an activity that is not inherent in the antibody itself.

As used herein, the term “percent occurrence of each amino acid residue at each position” refers to the percentage of instances in a sample in which an amino acid is found at a defined position within a particular sequence. For example, given the following three sequences:

KVR

KYP

KRP.

K occurs in position one in 100% of the instances and P occurs in position three in about 67% of the instances. In certain embodiments of the invention, the sequences selected for comparison are human immunoglobulin sequences.

As used herein, the term “most frequently occurring amino acids” at a specified position of a sequence in a population of polypeptides refers to the amino acid residues that have the highest percent occurrence at the indicated position in the indicated polypeptide population. For example, the most frequently occurring amino acids in each of the three most N-terminal positions in N1 sequences of CDRH3 sequences that are functionally expressed by human B cells are listed in Table 21, and the most frequently occurring amino acids in each of the three most N-terminal positions in N2 sequences of CDRH3 sequences that are functionally expressed by human B cells are listed in Table 22.

For the purposes of analyzing the occurrence of certain duplets (Example 13) and the information content (Example 14) of the libraries of the invention, and other libraries, a “central loop” of CDRH3 is defined. If the C-terminal 5 amino acids from Kabat CDRH3 (95-102) are removed, then the remaining sequence is termed the “central loop”. Thus, considering the duplet occurrence calculations of Example 13, using a CDRH3 of size 6 or less would not contribute to the analysis of the occurrence of duplets. A CDRH3 of size 7 would contribute only to the i-i+1 data set, a CDRH3 of size 8 would also contribute to the i-i+2 data set, and a CDRH3 of size 9 and larger would also contribute to the i-i+3 data set. For example, a CDR H3 of size 9 may have amino acids at positions 95-96-97-98-99-100-100A-101-102, but only the first four residues (bolded) would be part of the central loop and contribute to the pair-wise occurrence (duplet) statistics. As a further example, a CDRH3 of size 14 may have the sequence: 95-96-97-98-99-100-100A-100B-100C-100D-100E-100F-101-102. Here, only the first nine residues (bolded) contribute to the central loop.

Library screening requires a genotype-phenotype linkage. The term “genotype-phenotype linkage” is used in a manner consistent with its art-recognized meaning and refers to the fact that the nucleic acid (genotype) encoding a protein with a particular phenotype (e.g., binding an antigen) can be isolated from a library. For the purposes of illustration, an antibody fragment expressed on the surface of a phage can be isolated based on its binding to an antigen (e.g., Ladner et al.). The binding of the antibody to the antigen simultaneously enables the isolation of the phage containing the nucleic acid encoding the antibody fragment. Thus, the phenotype (antigen-binding characteristics of the antibody fragment) has been “linked” to the genotype (nucleic acid encoding the antibody fragment). Other methods of maintaining a genotype-phenotype linkage include those of Wittrup et al. (U.S. Pat. Nos. 6,300,065, 6,331,391, 6,423,538, 6,696,251, 6,699,658, and US Pub. No. 20040146976, each of which is incorporated by reference in its entirety), Miltenyi (U.S. Pat. No. 7,166,423, incorporated by reference in its entirety), Fandl (U.S. Pat. No. 6,919,183, US Pub No. 20060234311, each incorporated by reference in its entirety), Clausell-Tormos et al. (Chem. Biol., 2008, 15: 427, incorporated by reference in its entirety), Love et al. (Nat. Biotechnol., 2006, 24: 703, incorporated by reference in its entirety), and Kelly et al. (Chem. Commun., 2007, 14: 1773, incorporated by reference in its entirety). Any method which localizes the antibody protein with the gene encoding the antibody, in a way in which they can both be recovered while the linkage between them is maintained, is suitable.

2. Design of the Libraries

The antibody libraries of the invention are designed to reflect certain aspects of the preimmune repertoire as naturally created by the human immune system. Certain libraries of the invention are based on rational design informed by the collection of human V, D, and J genes, and other large databases of human heavy and light chain sequences (e.g., publicly known germline sequences; sequences from Jackson et al., J. Immunol. Methods, 2007, 324: 26, incorporated by reference in its entirety; sequences from Lee et al., Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety; and sequences compiled for rearranged VK and Vλ—see Appendices A and B filed herewith). Additional information may be found, for example, in Scaviner et al., Exp. Clin. Immunogenet., 1999, 16: 234; Tomlinson et al., J. Mol. Biol., 1992, 227: 799; and Matsuda et al., J. Exp. Med., 1998, 188: 2151 each incorporated by reference in its entirety. In certain embodiments of the invention, cassettes representing the possible V, D, and J diversity found in the human repertoire, as well as junctional diversity (i.e., N1 and N2), are synthesized de novo as single or double-stranded DNA oligonucleotides. In certain embodiments of the invention, oligonucleotide cassettes encoding CDR sequences are introduced into yeast along with one or more acceptor vectors containing heavy or light chain chassis sequences. No primer-based PCR amplification or template-directed cloning steps from mammalian cDNA or mRNA are employed. Through standard homologous recombination, the recipient yeast recombines the cassettes (e.g., CDR3s) with the acceptor vector(s) containing the chassis sequence(s) and constant regions, to create a properly ordered synthetic, full-length human heavy chain and/or light chain immunoglobulin library that can be genetically propagated, expressed, displayed, and screened. One of ordinary skill in the art will readily recognize that the chassis contained in the acceptor vector can be designed so as to produce constructs other than full-length human heavy chains and/or light chains. For example, in certain embodiments of the invention, the chassis may be designed to encode portions of a polypeptide encoding an antibody fragment or subunit of an antibody fragment, so that a sequence encoding an antibody fragment, or subunit thereof, is produced when the oligonucleotide cassette containing the CDR is recombined with the acceptor vector.

In certain embodiments, the invention provides a synthetic, preimmune human antibody repertoire comprising about 10⁷ to about 10²⁰ antibody members, wherein the repertoire comprises:

(a) selected human antibody heavy chain chassis (i.e., amino acids 1 to 94 of the heavy chain variable region, using Kabat's definition);

(b) a CDRH3 repertoire, designed based on the human IGHD and IGHJ germline sequences, the CDRH3 repertoire comprising the following:

-   -   (i) optionally, one or more tail regions;     -   (ii) one or more N1 regions, comprising about 0 to about 10         amino acids selected from the group consisting of fewer than 20         of the amino acid types preferentially encoded by the action of         terminal deoxynucleotidyl transferase (TdT) and functionally         expressed by human B cells;     -   (iii) one or DH segments, based on one or more selected IGHD         segments, and one or more N- or C-terminal truncations thereof;     -   (iv) one or more N2 regions, comprising about 0 to about 10         amino acids selected from the group consisting of fewer than 20         of the amino acids preferentially encoded by the activity of TdT         and functionally expressed by human B cells; and     -   (v) one or more H3-JH segments, based on one or more IGHJ         segments, and one or more N-terminal truncations thereof (e.g.,         down to XXWG);

(c) one or more selected human antibody kappa and/or lambda light chain chassis; and

(d) a CDRL3 repertoire designed based on the human IGLV and IGLJ germline sequences, wherein “L” may be a kappa or lambda light chain.

The heavy chain chassis may be any sequence with homology to Kabat residues 1 to 94 of an immunoglobulin heavy chain variable domain. Non-limiting examples of heavy chain chassis are included in the Examples, and one of ordinary skill in the art will readily recognize that the principles presented therein, and throughout the specification, may be used to derive additional heavy chain chassis.

As described above, the heavy chain chassis region is followed, optionally, by a “tail” region. The tail region comprises zero, one, or more amino acids that may or may not be selected on the basis of comparing naturally occurring heavy chain sequences. For example, in certain embodiments of the invention, heavy chain sequences available in the art may be compared, and the residues occurring most frequently in the tail position in the naturally occurring sequences included in the library (e.g., to produce sequences that most closely resemble human sequences). In other embodiments, amino acids that are used less frequently may be used. In still other embodiments, amino acids selected from any group of amino acids may be used. In certain embodiments of the invention, the length of the tail is zero (no residue) or one (e.g. G/D/E) amino acid. For the purposes of clarity, and without being bound by theory, in the naturally occurring human repertoire, the first ⅔ of the codon encoding the tail residue is provided by the FRM3 region of the VH gene. The amino acid at this position in naturally occurring heavy chain sequences may thus be considered to be partially encoded by the IGHV gene (⅔) and partially encoded by the CDRH3 (⅓). However, for the purposes of clearly illustrating certain aspects of the invention, the entire codon encoding the tail residue (and, therefore, the amino acid derived from it) is described herein as being part of the CDRH3 sequence.

As described above, there are two peptide segments derived from nucleotides which are added by TdT in the naturally occurring human antibody repertoire. These segments are designated N1 and N2 (referred to herein as N1 and N2 segments, domains, regions or sequences). In certain embodiments of the invention, N1 and N2 are about 0, 1, 2, or 3 amino acids in length. Without being bound by theory, it is thought that these lengths most closely mimic the N1 and N2 lengths found in the human repertoire (see FIG. 2). In other embodiments of the invention, N1 and N2 may be about 4, 5, 6, 7, 8, 9, or 10 amino acids in length. Similarly, the composition of the amino acid residues utilized to produce the N1 and N2 segments may also vary. In certain embodiments of the invention, the amino acids used to produce N1 and N2 segments may be selected from amongst the eight most frequently occurring amino acids in the N1 and N2 domains of the human repertoire (e.g., G, R, S, P, L, A, V, and T). In other embodiments of the invention, the amino acids used to produce the N1 and N2 segments may be selected from the group consisting of fewer than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 of the amino acids preferentially encoded by the activity of TdT and functionally expressed by human B cells. Alternatively, N1 and N2 may comprise amino acids selected from any group of amino acids. It is not required that N1 and N2 be of a similar length or composition, and independent variation of the length and composition of N1 and N2 is one method by which additional diversity may be introduced into the library.

The DH segments of the libraries are based on the peptides encoded by the naturally occurring IGHD gene repertoire, with progressive deletion of residues at the N- and C-termini. IGHD genes may be read in multiple reading frames, and peptides representing these reading frames, and their N- and C-terminal deletions are also included in the libraries of the invention. In certain embodiments of the invention, DH segments as short as three amino acid residues may be included in the libraries. In other embodiments of the invention, DH segments as short as about 1, 2, 4, 5, 6, 7, or 8 amino acids may be included in the libraries.

The H3-JH segments of the libraries are based on the peptides encoded by the naturally occurring IGHJ gene repertoire, with progressive deletion of residues at the N-terminus. The N-terminal portion of the IGHJ segment that makes up part of the CDRH3 is referred to herein as H3-JH. In certain embodiments of the invention, the H3-JH segment may be represented by progressive N-terminal deletions of one or more H3-JH residues, down to two H3-JH residues. In other embodiments of the invention, the H3-JH segments of the library may contain N-terminal deletions (or no deletions) down to about 6, 5, 4, 3, 2, 1, or 0 H3-JH residues.

The light chain chassis of the libraries may be any sequence with homology to Kabat residues 1 to 88 of naturally occurring light chain (κ or λ) sequences. In certain embodiments of the invention, the light chain chassis of the invention are synthesized in combinatorial fashion, utilizing VL and JL segments, to produce one or more libraries of light chain sequences with diversity in the chassis and CDR3 sequences. In other embodiments of the invention, the light chain CDR3 sequences are synthesized using degenerate oligonucleotides or trinucleotides and recombined with the light chain chassis and light chain constant region, to form full-length light chains.

The instant invention also provides methods for producing and using such libraries, as well as libraries comprising one or more immunoglobulin domains or antibody fragments. Design and synthesis of each component of the claimed antibody libraries is provided in more detail below.

2.1. Design of the Antibody Library Chassis Sequences

One step in building certain libraries of the invention is the selection of chassis sequences, which are based on naturally occurring variable domain sequences (e.g., IGHV and IGLV). This selection can be done arbitrarily, or by the selection of chassis that meet certain criteria. For example, the Kabat database, an electronic database containing non-redundant rearranged antibody sequences, can be queried for those heavy and light chain germline sequences that are most frequently represented. The BLAST search algorithm, or more specialized tools such as SoDA (Volpe et al., Bioinformatics, 2006, 22: 438-44, incorporated by reference in its entirety), can be used to compare rearranged antibody sequences with germline sequences, using the V BASE2 database (Retter et al., Nucleic Acids Res., 2005, 33: D671-D674), or similar collections of human V, D, and J genes, to identify the germline families that are most frequently used to generate functional antibodies.

Several criteria can be utilized for the selection of chassis for inclusion in the libraries of the invention. For example, sequences that are known (or have been determined) to express poorly in yeast, or other organisms used in the invention (e.g., bacteria, mammalian cells, fungi, or plants) can be excluded from the libraries. Chassis may also be chosen based on their representation in the peripheral blood of humans. In certain embodiments of the invention, it may be desirable to select chassis that correspond to germline sequences that are highly represented in the peripheral blood of humans. In other embodiments, it may be desirable to select chassis that correspond to germline sequences that are less frequently represented, for example, to increase the canonical diversity of the library. Therefore, chassis may be selected to produce libraries that represent the largest and most structurally diverse group of functional human antibodies. In other embodiments of the invention, less diverse chassis may be utilized, for example, if it is desirable to produce a smaller, more focused library with less chassis variability and greater CDR variability. In some embodiments of the invention, chassis may be selected based on both their expression in a cell of the invention (e.g., a yeast cell) and the diversity of canonical structures represented by the selected sequences. One may therefore produce a library with a diversity of canonical structures that express well in a cell of the invention.

2.1.1. Design of the Heavy Chain Chassis Sequences

In certain embodiments of the invention, the antibody library comprises variable heavy domains and variable light domains, or portions thereof. Each of these domains is built from certain components, which will be more fully described in the examples provided herein. In certain embodiments, the libraries described herein may be used to isolate fully human antibodies that can be used as diagnostics and/or therapeutics. Without being bound by theory, antibodies with sequences most similar or identical to those most frequently found in peripheral blood (for example, in humans) may be less likely to be immunogenic when administered as therapeutic agents.

Without being bound by theory, and for the purposes of illustrating certain embodiments of the invention, the VH domains of the library may be considered to comprise three primary components: (1) a VH “chassis”, which includes amino acids 1 to 94 (using Kabat numbering), (2) the CDRH3, which is defined herein to include the Kabat CDRH3 proper (positions 95-102), and (3) the FRM4 region, including amino acids 103 to 113 (Kabat numbering). The overall VH structure may therefore be depicted schematically (not to scale) as:

The selection and design of VH chassis sequences based on the human IGHV germline repertoire will become more apparent upon review of the examples provided herein. In certain embodiments of the invention, the VH chassis sequences selected for use in the library may correspond to all functionally expressed human IGHV germline sequences. Alternatively, IGHV germline sequences may be selected for representation in a library according to one or more criteria. For example, in certain embodiments of the invention, the selected IGHV germline sequences may be among those that are most highly represented among antibody molecules isolated from the peripheral blood of healthy adults, children, or fetuses.

In certain embodiments, it may be desirable to base the design of the VH chassis on the utilization of IGHV germline sequences in adults, children, or fetuses with a disease, for example, an autoimmune disease. Without being bound by theory, it is possible that analysis of germline sequence usage in the antibody molecules isolated from the peripheral blood of individuals with autoimmune disease may provide information useful for the design of antibodies recognizing human antigens.

In some embodiments, the selection of IGHV germline sequences for representation in a library of the invention may be based on their frequency of occurrence in the peripheral blood. For the purposes of illustration, four IGHV1 germline sequences (IGHV1-2, IGHV1-18, IGHV1-46, and IGHV1-69) comprise about 80% of the IGHV1 family repertoire in peripheral blood. Thus, the specific IGHV1 germline sequences selected for representation in the library may include those that are most frequently occurring and that cumulatively comprise at least about 80% of the IGHV1 family repertoire found in peripheral blood. An analogous approach can be used to select specific IGHV germline sequences from any other IGHV family (i.e., IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6, and IGHV7). The specific germline sequences chosen for representation of a particular IGHV family in a library of the invention may therefore comprise at least about 100%, 99%, 98%, 97%, 96% 95%, 94%, 93%, 92%, 91% 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the particular IGHV family member repertoire found in peripheral blood.

In some embodiments, the selected IGHV germline sequences may be chosen to maximize the structural diversity of the VH chassis library. Structural diversity may be evaluated by, for example, comparing the lengths, compositions, and canonical structures of CDRH1 and CDRH2 in the IGHV germline sequences. In human IGHV sequences, the CDRH1 (Kabat definition) may have a length of 5, 6 or 7 amino acids, while CDRH2 (Kabat definition) may have length of 16, 17, 18 or 19 amino acids. The amino acid compositions of the IGHV germline sequences and, in particular, the CDR domains, may be evaluated by sequence alignments, as presented in the Examples. Canonical structure may be assigned, for example, according to the methods described by Chothia et al., J. Mol. Biol., 1992, 227: 799, incorporated by reference in its entirety.

In certain embodiments of the invention, it may be advantageous to design VH chassis based on IGHV germline sequences that may maximize the probability of isolating an antibody with particular characteristics. For example, without being bound by theory, in some embodiments it may be advantageous to restrict the IGHV germline sequences to include only those germline sequences that are utilized in antibodies undergoing clinical development, or antibodies that have been approved as therapeutics. On the other hand, in some embodiments, it may be advantageous to produce libraries containing VH chassis that are not represented amongst clinically utilized antibodies. Such libraries may be capable of yielding antibodies with novel properties that are advantageous over those obtained with the use of “typical” IGHV germline sequences, or enabling studies of the structures and properties of “atypical” IGHV germline sequences or canonical structures.

One of ordinary skill in the art will readily recognize that a variety of other criteria can be used to select IGHV germline sequences for representation in a library of the invention. Any of the criteria described herein may also be combined with any other criteria. Further exemplary criteria include the ability to be expressed at sufficient levels in certain cell culture systems, solubility in particular antibody formats (e.g., whole immunoglobulins and antibody fragments), and the thermodynamic stability of the individual domains, whole immunoglobulins, or antibody fragments. The methods of the invention may be applied to select any IGHV germline sequence that has utility in an antibody library of the instant invention.

In certain embodiments of the invention, the VH chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 94 of one or more of the following IGHV germline sequences: IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B, IGHV5-51, IGHV6-1, and IGHV7-4-1. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

In other embodiments, the VH chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 94 of the following IGHV germline sequences: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV3-7, IGHV3-15, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-48, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B, and IGHV5-51. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences. The amino acid sequences of these chassis are presented in Table 5.

2.1.1.1. Heavy Chain Chassis Variants

While the selection of the VH chassis with sequences based on the IGHV germline sequences is expected to support a large diversity of CDRH3 sequences, further diversity in the VH chassis may be generated by altering the amino acid residues comprising the CDRH1 and/or CDRH2 regions of each chassis selected for inclusion in the library (see Example 2).

In certain embodiments of the invention, the alterations or mutations in the amino acid residues comprising the CDRH1 and CDRH2 regions, or other regions, of the IGHV germline sequences are made after analyzing the sequence identity within data sets of rearranged human heavy chain sequences that have been classified according to the identity of the original IGHV germline sequence from which the rearranged sequences are derived. For example, from a set of rearranged antibody sequences, the IGHV germline sequence of each antibody is determined, and the rearranged sequences are classified according to the IGHV germline sequence. This determination is made on the basis of sequence identity.

Next, the occurrence of any of the 20 amino acid residues at each position in these sequences is determined. In certain embodiments of the invention, one may be particularly interested in the occurrence of different amino acid residues at the positions within CDRH1 and CDRH2, for example if increasing the diversity of the antigen-binding portion of the VH chassis is desired. In other embodiments of the invention, it may be desirable to evaluate the occurrence of different amino acid residues in the framework regions. Without being bound by theory, alterations in the framework regions may impact antigen binding by altering the spatial orientation of the CDRs.

After the occurrence of amino acids at each position of interest has been identified, alterations may be made in the VH chassis sequence, according to certain criteria. In some embodiments, the objective may be to produce additional VH chassis with sequence variability that mimics the variability observed in the heavy chain domains of rearranged human antibody sequences (derived from respective IGHV germline sequences) as closely as possible, thereby potentially obtaining sequences that are most human in nature (i.e., sequences that most closely mimic the composition and length of human sequences). In this case, one may synthesize additional VH chassis sequences that include mutations naturally found at a particular position and include one or more of these VH chassis sequences in a library of the invention, for example, at a frequency that mimics the frequency found in nature. In another embodiment of the invention, one may wish to include VH chassis that represent only mutations that most frequently occur at a given position in rearranged human antibody sequences. For example, rather than mimicking the human variability precisely, as described above, and with reference to exemplary Tables 6 and 7, one may choose to include only top 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, amino acid residues that most frequently occur at each position. For the purposes of illustration, and with reference to Table 6, if one wished to include the top four most frequently occurring amino acid residues at position 31 of the VH1-69 sequence, then position 31 in the VH1-69 sequence would be varied to include S, N, T, and R. Without being bound by theory, it is thought that the introduction of diversity by mimicking the naturally occurring composition of the rearranged heavy chain sequences is likely to produce antibodies that are most human in composition. However, the libraries of the invention are not limited to heavy chain sequences that are diversified by this method, and any criteria can be used to introduce diversity into the heavy chain chassis, including random or rational mutagenesis. For example, in certain embodiments of the invention, it may be preferable to substitute neutral and/or smaller amino acid residues for those residues that occur in the IGHV germline sequence. Without being bound by theory, neutral and/or smaller amino acid residues may provide a more flexible and less sterically hindered context for the display of a diversity of CDR sequences.

Example 2 illustrates the application of this method to heavy chains derived from a particular IGHV germline. One of ordinary skill in the art will readily recognize that this method can be applied to any germline sequence, and can be used to generate at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000, 10⁴, 10⁵, 10⁶, or more variants of each heavy chain chassis.

2.1.2. Design of the Light Chain Chassis Sequences

The light chain chassis of the invention may be based on kappa and/or lambda light chain sequences. The principles underlying the selection of light chain variable (IGLV) germline sequences for representation in the library are analogous to those employed for the selection of the heavy chain sequences (described above and in Examples 1 and 2). Similarly, the methods used to introduce variability into the selected heavy chain chassis may also be used to introduce variability into the light chain chassis.

Without being bound by theory, and for the purposes of illustrating certain embodiments of the invention, the VL domains of the library may be considered to comprise three primary components: (1) a VL “chassis”, which includes amino acids 1 to 88 (using Kabat numbering), (2) the VLCDR3, which is defined herein to include the Kabat CDRL3 proper (positions 89-97), and (3) the FRM4 region, including amino acids 98 to 107 (Kabat numbering). The overall VL structure may therefore be depicted schematically (not to scale) as:

In certain embodiments of the invention, the VL chassis of the libraries include one or more chassis based on IGKV germline sequences. In certain embodiments of the invention, the VL chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 88 of one or more of the following IGKV germline sequences: IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

In other embodiments, the VL chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 88 of the following IGKV germline sequences: IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, IGKV1-39, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, and IGKV4-1. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences. The amino acid sequences of these chassis are presented in Table 11.

In certain embodiments of the invention, the VL chassis of the libraries include one or more chassis based on IGλV germline sequences. In certain embodiments of the invention, the VL chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 88 of one or more of the following IGλV germline sequences:

IGλV3-1, IGλV3-21, IGλV2-14, IGλV1-40, IGλV3-19, IGλV1-51, IGλV1-44, IGλV6-57, IGλV2-8, IGλV3-25, IGλV2-23, IGλV3-10, IGλV4-69, IGλV1-47, IGλV2-11, IGλV7-43, IGλV7-46, IGλV5-45, IGλV4-60, IGλV10-54, IGλV8-61, IGλV3-9, IGλV1-36, IGλV2-18, IGλV3-16, IGλV3-27, IGλV4-3, IGλV5-39, IGλV9-49, and IGλV3-12. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

In other embodiments, the VL chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 88 of the following IGλV germline sequences: IGλV3-1, IGλV3-21, IGλV2-14, IGλV1-40, IGλV3-19, IGλV1-51, IGλV1-44, IGλV6-57, IGλV4-69, IGλV7-43, and IGλV5-45. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences. The amino acid sequences of these chassis are presented in Table 14.

2.2. Design of the Antibody Library CDRH3 Components

It is known in the art that diversity in the CDR3 region of the heavy chain is sufficient for most antibody specificities (Xu and Davis, Immunity, 2000, 13: 27-45, incorporated by reference in its entirety) and that existing successful libraries have been created using CDRH3 as the major source of diversification (Hoogenboom et al., J. Mol. Biol., 1992, 227: 381; Lee et al., J. Mol. Biol., 2004, 340: 1073 each of which is incorporated by reference in its entirety). It is also known that both the DH region and the N1/N2 regions contribute to the CDRH3 functional diversity (Schroeder et al., J. Immunol., 2005, 174: 7773 and Mathis et al., Eur J. Immunol., 1995, 25: 3115, each of which is incorporated by reference in its entirety). For the purposes of the present invention, the CDHR3 region of naturally occurring human antibodies can be divided into five segments: (1) the tail segment, (2) the N1 segment, (3) the DH segment, (4) the N2 segment, and (5) the JH segment. As exemplified below, the tail, N1 and N2 segments may or may not be present.

In certain embodiments of the invention, the method for selecting amino acid sequences for the synthetic CDRH3 libraries includes a frequency analysis and the generation of the corresponding variability profiles of existing rearranged antibody sequences. In this process, which is described in more detail in the Examples section, the frequency of occurrence of a particular amino acid residue at a particular position within rearranged CDRH3s (or any other heavy or light chain region) is determined. Amino acids that are used more frequently in nature may then be chosen for inclusion in a library of the invention.

2.2.1. Design and Selection of the DH Segment Repertoire

In certain embodiments of the invention, the libraries contain CDRH3 regions comprising one or more segments designed based on the IGHD gene germline repertoire. In some embodiments of the invention, DH segments selected for inclusion in the library are selected and designed based on the most frequent usage of human IGHD genes, and progressive N-terminal and C-terminal deletions thereof, to mimic the in vivo processing of the IGHD gene segments. In some embodiments of the invention, the DH segments of the library are about 3 to about 10 amino acids in length. In some embodiments of the invention, the DH segments of the library are about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, or a combination thereof. In certain embodiments, the libraries of the invention may contain DH segments with a wide distribution of lengths (e.g., about 0 to about 10 amino acids). In other embodiments, the length distribution of the DH may be restricted (e.g., about 1 to about 5 amino acids, about 3 amino acids, about 3 and about 5 amino acids, and so on). In certain embodiments of the library, the shortest DH segments may be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In certain embodiments of the invention, libraries may contain DH segments representative of any reading frame of any IGHD germline sequence. In certain embodiments of the invention, the DH segments selected for inclusion in a library include one or more of the following IGHD sequences, or their derivatives (i.e., any reading frame and any degree of N-terminal and C-terminal truncation): IGHD3-10, IGHD3-22, IGHD6-19, IGHD6-13, IGHD3-3, IGHD2-2, IGHD4-17, IGHD1-26, IGHD5-5/5-18, IGHD2-15, IGHD6-6, IGHD3-9, IGHD5-12, IGHD5-24, IGHD2-21, IGHD3-16, IGHD4-23, IGHD1-1, IGHD1-7, IGHD4-4/4-11, IGHD1-20, IGHD7-27, IGHD2-8, and IGHD6-25. In some embodiments of the invention, a library may contain one or more of these sequences, allelic variants thereof, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

For the purposes of illustration, progressive N-terminal and C-terminal deletions of IGHD3-10, reading frame 1, are enumerated in the Table 1. N-terminal and C-terminal deletions of other IGHD sequences and reading frames are also encompassed by the invention, and one of ordinary skill in the art can readily determine these sequences using, for example, the non-limiting exemplary data presented in Table 16. and/or the methods outlined above. Table 18 (Example 5) enumerates certain DH segments used in certain embodiments of the invention.

TABLE 1 Example of Progressive N- and C-terminal Deletions of Reading Frame 1 for Gene IGHD3-10, Yielding DH Segments DH SEQ ID NO: DH SEQ ID NO: VLLWFGELL 1 LWFGEL (a) VLLWFGEL (a) LWFGE (a) VLLWFGE (a) LWFG (a) VLLLWFG (a) LWF (a) VLLWF (a) WFGELL (a) VLLW (a) WFGEL (a) VLL (a) WFGE (a) LLWFGELL (a) WFG (a) LLWFGEL (a) FGELL (a) LLWFGE (a) FGEL (a) LLWFG (a) FGE (a) LLWF (a) GELL (a) LLW (a) GEL (a) LWFGELL (a) ELL (a) (a): derived from SEQ ID NO: 1.

In certain embodiments of the invention, the DH segments selected for inclusion in a library include one or more of the following IGHD sequences, or their derivatives (i.e., any reading frame and any degree N-terminal and C-terminal truncation): IGHD3-10, IGHD3-22, IGHD6-19, IGHD6-13, IGHD3-03, IGHD2-02, IGHD4-17, IGHD1-26, IGHD5-5/5-18, and IGHD2-15. In some embodiments of the invention, a library may contain one or more of these sequences, allelic variants thereof, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

In certain embodiments of the invention, the DH segments selected for inclusion in a library include one or more of the following IGHD sequences, wherein the notation “_x” denotes the reading frame of the gene, or their derivatives (i.e., any degree of N-terminal or C-terminal truncation): IGHD1-26_(—)1, IGHD1-26_(—)3, IGHD2-2_(—)2, IGHD2-2_(—)3, IGHD2-15_(—)2, IGHD3-3_(—)3, IGHD3-10_(—)1, IGHD3-10_(—)2, IGHD3-10_(—)3, IGHD3-22_(—)2, IGHD4-17_(—)2, IGHD5-5_(—)3, IGHD6-13_(—)1, IGHD6-13_(—)2, IGHD6-19_(—)1, and IGHD6-19_(—)2. In some embodiments of the invention, a library may contain one or more of these sequences, allelic variants thereof, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

In certain embodiments of the invention, the libraries are designed to reflect a pre-determined length distribution of N- and C-terminal deleted IGHD segments. For example, in certain embodiments of the library, the DH segments of the library may be designed to mimic the natural length distribution of DH segments found in the human repertoire. For example, the relative occurrence of different IGHD segments in rearranged human antibody heavy chain domains from Lee et al. (Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety). Table 2 shows the relative occurrence of the top 68% of IGHD segments from Lee et al.

TABLE 2 Relative Occurrence of Top 68% of IGHD Gene Usage from Lee et al. IGHD Sequence SEQ ID Relative Reading Frame (Parent) NO: Occurrence IGHD3-10_1 VLLWFGELL 1 4.3% IGHD3-10_2 YYYGSGSYYN 2 8.4% IGHD3-10_3 ITMVRGVII 3 4.0% IGHD3-22_2 YYYDSSGYYY 4 15.6%  IGHD6-19_1 GYSSGWY 5 7.4% IGHD6-19_2 GIAVAG 6 6.0% IGHD6-13_1 GYSSSWY 7 8.4% IGHD6-13_2 GIAAAG 8 5.3% IGHD3-3_3 ITIFGVVII 9 7.4% IGHD2-2_2 GYCSSTSCYT 10 5.2% IGHD2-2_3 DIVVVPAAM 11 4.1% IGHD4-17_2 DYGDY 12 6.8% IGHD1-26_1 GIVGATT 13 2.9% IGHD1-26_3 YSGSYY 14 4.3% IGHD5-5_3 GYSYGY 15 4.3% IGHD2-15_2 GYCSGGSCYS 16 5.6%

In certain embodiments, these relative occurrences may be used to design a library with DH prevalence that is similar to the IGHD usage found in peripheral blood. In other embodiments of the invention, it may be preferable to bias the library toward longer or shorter DH segments, or DH segments of a particular composition. In other embodiments, it may be desirable to use all DH segments selected for the library in equal proportion.

In certain embodiments of the invention, the most commonly used reading-frames of the ten most frequently occurring IGHD sequences are utilized, and progressive N-terminal and C-terminal deletions of these sequences are made, thus providing a total of 278 non-redundant DH segments that are used to create a CDRH3 repertoire of the instant invention (Table 18). In some embodiments of the invention, the methods described above can be applied to produce libraries comprising the top 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 expressed IGHD sequences, and progressive N-terminal and C-terminal deletions thereof. As with all other components of the library, while the DH segments may be selected from among those that are commonly expressed, it is also within the scope of the invention to select these gene segments based on the fact that they are less commonly expressed. This may be advantageous, for example, in obtaining antibodies toward self-antigens or in further expanding the diversity of the library. Alternatively, DH segments can be used to add compositional diversity in a manner that is strictly relative to their occurrence in actual human heavy chain sequences.

In certain embodiments of the invention, the progressive deletion of IGHD genes containing disulfide loop encoding segments may be limited, so as to leave the loop intact and to avoid the presence of unpaired cysteine residues. In other embodiments of the invention, the presence of the loop can be ignored and the progressive deletion of the IGHD gene segments can occur as for any other segments, regardless of the presence of unpaired cysteine residues. In still other embodiments of the invention, the cysteine residues can be mutated to any other amino acid.

2.2.1.2 Design and Selection of DH Segments from Non-Human Vertebrates

In certain embodiment of the invention, DH segments from non-human vertebrates may be used in conjunction with human VH, N1, N2, and H3-JH segments to produce CDRH3s and/or antibodies in which all segments except the DH segment are synthesized with reference to human sequences. Without being bound by theory, it is anticipated that the extensive variability in the DH segment of antibodies, for example as the result of somatic hypermutation, may make this region more permissive to the inclusion of sequences that have non-human characteristics, without sacrificing the ability to recognize a broad variety of antigens or introducing immunogenic sequences.

The general methods taught herein are readily applicable to information derived from species other than humans. Example 16 presents exemplary DH segments from a variety of species and outlines methods for their inclusion in the libraries of the invention. These methods may be readily applied to information derived from other species and/or sources of information other than those presented in Example 16. For example, as IGHD sequence data becomes available for additional species (e.g., as a result of focused sequencing efforts), one of ordinary skill in the art could use the teachings of this application to construct libraries with DH segments derived from these species.

In certain embodiments of the invention, a library may contain one or more DH segments derived from the IGHD genes presented in Table 55. As further enumerated in Example 16, these sequences can be selected according to one or more non-limiting criteria, including diversity in length and sequence, maximal (or minimal) human “string content,” and/or the absence or minimization of T cell epitopes. Like the human IGHD sequences discussed elsewhere in the application, the non-human IGHD segments of the invention may be deleted at their N- and/or C-termini to provide DH segments with a minimal length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The length distribution, reading frame, and frequency of inclusion of the non-human DH segments selected for inclusion in the library may be varied as presented for the human DH segments. Non-human DH segments include those derived from non-human IGHD genes according to the methods presented herein, allelic variants thereof, and amino acid and nucleotide sequence at least about 99.9% , 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

IGHD segments may be obtained from multiple species, including camel, shark, mouse, rat, llama, fish, rabbit, and so on. Non-limiting exemplary species from which IGHD segments may be obtained include Mus musculus, Camelus sp., Llama sp., Camelidae sp., Raja sp., Ginglymostoma sp., Carcharhinus sp., Heterodontus sp., Hydrolagus sp., Ictalurus sp., Gallus sp., Bos sp., Marmaronetta sp., Aythya sp., Netta sp., Equus sp., Pentalagus sp., Bunolagus sp., Nesolagus sp., Romerolagus sp., Brachylagus sp., Sylvilagus sp., Oryctolagus, sp., Poelagus sp., Ovis sp., Sus sp., Gadus sp., Salmo sp., Oncorhynchus sp, Macaca sp., Rattus sp., Pan sp., Hexanchus sp., Heptranchias sp., Notorynchus sp., Chlamydoselachus sp., Heterodontus sp. Pristiophorus sp., Pliotrema sp., Squatina sp., Carcharia sp., Mitsukurina sp., Lamma sp., Isurus sp., Carcharodon sp., Cetorhinus sp., Alopias sp., Nebrius sp., Stegostoma sp., Orectolobus sp., Eucrossorhinus sp., Sutorectus sp., Chiloscyllium sp., Hemiscyllium sp., Brachaelurus sp., Heteroscyllium sp., Cirrhoscyllium sp., Parascyllium sp., Rhincodon sp., Apristurus sp., Atelomycterus sp., Cephaloscyllium sp., Cephalurus sp., Dichichthys sp., Galeus sp., Halaelurus sp., Haploblepharus sp., Parmaturus sp., Pentanchus sp., Poroderna sp., Schroederichthys sp., Scyliorhinus sp., Pseudotriakis sp., Scylliogaleus sp., Furgaleus sp., Hemitriakis sp., Mustelus sp., Triakis sp., Iago sp., Galeorhinus sp., Hypogaleus sp., Chaenogaleus sp., Hemigaleus sp., Paragaleus sp., Galeocerdo sp., Prionace sp., Sciolodon sp., Loxodon sp., Rhizoprionodon sp., Aprionodon sp., Negaprion sp., Hypoprion sp., Carcharhinus sp., Isogomphodon sp., Triaenodon sp., Sphyrna sp., Echinorhinus sp., Oxynotus sp., Squalus sp., Centroscyllium sp., Etmopterus sp., Centrophorus sp., Cirrhigaleus sp., Deania sp., Centroscymnus sp., Scymnodon sp., Dalatias sp., Euprotomicrus sp., Isistius sp., Squaliolus sp., Heteroscymnoides sp., Somniosus sp. and Megachasma sp.

Publications discussing IGHD segments from additional species and/or methods of obtaining such segments include, for example, Ye, Immunogenetics, 2004, 56: 399; De Genst et al., Dev. Comp. Immunol., 2006, 30: 187; Dooley and Flajnik, Dev. Comp. Immunol. 2006, 30: 43; Bengten et al., Dev. Comp. Immunol., 2006, 30: 77; Ratcliffe,

Dev. Comp. Immunol., 2006, 30: 101; Zhao et al., Dev. Comp. Immunol., 2006, 30: 175; Lundqvist et al., Dev. Comp. Immunol., 2006, 30: 93; Wagner, Dev. Comp. Immunol. 2006, 30: 155; Mage et al., Dev. Comp. Immunol., 2006, 30: 137; Malecek et al., J. Immunol., 2005, 175: 8105; Jenne et al., Dev. Comp. Immunol., 2006, 30: 165; Butler et al., Dev. Comp. Immunol., 2006, 30: 199; Solem et al., Dev. Comp. Immunol., 2006, 30: 57; Das et al., Immunogenetics, 2008, 60: 47, and Kiss et al.,

Nucleic Acids Res., 2006, 34: e132, each of which is incorporated by reference in its entirety.

Given the degree of variability in N1 and N², these segments might also be considered possible regions for substitution with non-human sequences, that is, sequences with composition biases not arising from those of human terminal deoxynucleotide transferase. The methods taught herein for the identification and analysis of the N1 and N2 regions of human antibodies are also readily applicable to non-human antibodies.

2.2.2. Design and Selection of the H3-JH Segment Repertoire

There are six IGHJ (joining) segments, IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6. The amino acid sequences of the parent segments and the progressive N-terminal deletions are presented in Table 20 (Example 5). Similar to the N- and C-terminal deletions that the IGHD genes undergo, natural variation is introduced into the IGHJ genes by N-terminal “nibbling”, or progressive deletion, of one or more codons by exonuclease activity.

The H3-JH segment refers to the portion of the IGHJ segment that is part of CDRH3. In certain embodiments of the invention, the H3-JH segment of a library comprises one or more of the following sequences: AEYFQH (SEQ ID NO: 17), EYFQH, YFQH, FQH, QH, H, YWYFDL (SEQ ID NO: 18), WYFDL, YFDL, FDL, DL, L, AFDV (SEQ ID NO: 19), FDV, DV, V, YFDY (SEQ ID NO: 20), FDY, DY, Y, NWFDS (SEQ ID NO: 21), WFDS, FDS, DS, S, YYYYYGMDV (SEQ ID NO: 22), YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, MDV, and DV. In some embodiments of the invention, a library may contain one or more of these sequences, allelic variations thereof, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 60%, 55%, or 50% identical to one or more of these sequences.

In other embodiments of the invention, the H3-JH segment may comprise about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acids. For example, the H3-JH segment of JH1_(—)4 (Table 20) has a length of three residues, while non-deleted JH6 has an H3-JH segment length of nine residues. The FRM4-JH region of the IGHJ segment begins with the sequence WG(Q/R)G (SEQ ID NO: 23) and corresponds to the portion of the IGHJ segment that makes up part of framework 4. In certain embodiments of the invention, as enumerated in Table 20, there are 28 H3-JH segments that are included in a library. In certain other embodiments, libraries may be produced by utilizing about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the IGHJ segments enumerated above or in Table 20.

2.2.3. Design and Selection of the N1 and N2 Segment Repertoires

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved enzyme from vertebrates that catalyzes the attachment of 5′ triphosphates to the 3′ hydroxyl group of single- or double-stranded DNA. Hence, the enzyme acts as a template-independent polymerase (Koiwai et al., Nucleic Acids Res., 1986, 14: 5777; Basu et al., Biochem. Biophys. Res. Comm., 1983, 111: 1105, each incorporated by reference in its entirety). In vivo, TdT is responsible for the addition of nucleotides to the V-D and D-J junctions of antibody heavy chains (Alt and Baltimore, PNAS, 1982, 79: 4118; Collins et al., J. Immunol., 2004, 172: 340, each incorporated by reference in its entirety). Specifically, TdT is responsible for creating the N1 and N2 (non-templated) segments that flank the D (diversity) region.

In certain embodiments of the invention, the length and composition of the N1 and N2 segments are designed rationally, according to statistical biases in amino acid usage found in naturally occurring N1 and N2 segments in human antibodies. One embodiment of a library produced via this method is described in Example 5. According to data compiled from human databases (Jackson et al., J. Immunol. Methods, 2007, 324: 26, incorporated by reference in its entirety), there are an average of 3.02 amino acid insertions for N1 and 2.4 amino acid insertions for N2, not taking into account insertions of two nucleotides or less (FIG. 2). In certain embodiments of the invention, N1 and N2 segments are restricted to lengths of zero to three amino acids. In other embodiments of the invention, N1 and N2 may be restricted to lengths of less than about 4, 5, 6, 7, 8, 9, or 10 amino acids.

In some embodiments of the invention, the composition of these sequences may be chosen according to the frequency of occurrence of particular amino acids in the N1 and N2 sequences of natural human antibodies (for examples of this analysis, see, Tables 21 to 23, in Example 5). In certain embodiments of the invention, the eight most commonly occurring amino acids in these regions (i.e., G, R, S, P, L, A, T, and V) are used to design the synthetic N1 and N2 segments. In other embodiments of the invention about the most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 most commonly occurring amino acids may be used in the design of the synthetic N1 and N2 segments. In still other embodiments, all 20 amino acids may be used in these segments. Finally, while it is possible to base the designed composition of the N1 and N2 segments of the invention on the composition of naturally occurring N1 and N2 segments, this is not a requirement. The N1 and N2 segments may comprise amino acids selected from any group of amino acids, or designed according to other criteria considered for the design of a library of the invention. A person of ordinary skill in the art would readily recognize that the criteria used to design any portion of a library of the invention may vary depending on the application of the particular library. It is an object of the invention that it may be possible to produce a functional library through the use of N1 and N2 segments selected from any group of amino acids, no N1 or N2 segments, or the use of N1 and N2 segments with compositions other than those described herein.

One important difference between the libraries of the current invention and other libraries known in the art is the consideration of the composition of naturally occurring duplet and triplet amino acid sequences during the design of the library. Table 23 shows the top twenty-five naturally occurring duplets in the N1 and N2 regions. Many of these can be represented by the general formula (G/P)(G/R/S/P/L/A/V/T) or (R/S/L/A/V/T)(G/P). In certain embodiments of the invention, the synthetic N1 and N2 regions may comprise all of these duplets. In other embodiments, the library may comprise the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 most common naturally occurring N1 and/or N2 duplets. In other embodiments of the invention, the libraries may include duplets that are less frequently occurring (i.e., outside of the top 25). The composition of these additional duplets or triplets could readily be determined, given the methods taught herein.

Finally, the data from the naturally occurring triplet N1 and N2 regions demonstrates that the naturally occurring N1 and N2 triplet sequences can often be represented by the formulas (G)(G)(G/R/S/P/L/A/V/T), (G)(R/S/P/L/A/V/T)(G), or (R/S/P/L/A/V/T)(G)(G). In certain embodiments of the invention, the library may comprise the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 most commonly occurring N1 and/or N2 triplets. In other embodiments of the invention, the libraries may include triplets that are less frequently occurring (i.e., outside of the top 25). The composition of these additional duplets or triplets could readily be determined, given the methods taught herein.

In certain embodiments of the invention, there are about 59 total N1 segments and about 59 total N2 segments used to create a library of CDRH3s. In other embodiments of the invention, the number of N1 segments, N2 segments, or both is increased to about 141 (see, for example, Example 5). In other embodiments of the invention, one may select a total of about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 1000, 10⁴, or more N1 and/or N2 segments for inclusion in a library of the invention.

One of ordinary skill in the art will readily recognize that, given the teachings of the instant specification, it is well within the realm of normal experimentation to extend the analysis detailed herein, for example, to generate additional rankings of naturally occurring duplet and triplet (or higher order) N regions that extend beyond those presented herein (e.g., using sequence alignment, the SoDA algorithm, and any database of human sequences (Volpe et al., Bioinformatics, 2006, 22: 438-44, incorporated by reference in its entirety). An ordinarily skilled artisan would also recognize that, based on the information taught herein, it is now possible to produce libraries that are more diverse or less diverse (i.e., more focused) by varying the number of distinct amino acid sequences used in the N1 pool and/or N2 pool.

As described above, many alternative embodiments are envisioned, in which the compositions and lengths of the N1 and N2 segments vary from those presented in the Examples herein. In some embodiments, sub-stoichiometric synthesis of trinucleotides may be used for the synthesis of N1 and N2 segments. Sub-stoichiometric synthesis with trinucleotides is described in Knappik et al. (U.S. Pat. No. 6,300,064, incorporated by reference in its entirety). The use of sub-stoichiometric synthesis would enable synthesis with consideration of the length variation in the N1 and N2 sequences.

In addition to the embodiments described above, a model of the activity of TdT may also be used to determine the composition of the N1 and N2 sequences in a library of the invention. For example, it has been proposed that the probability of incorporating a particular nucleotide base (A, C, G, T) on a polynucleotide, by the activity of TdT, is dependent on the type of base and the base that occurs on the strand directly preceding the base to be added. Jackson et al., (J. Immunol. Methods, 2007, 324: 26, incorporated by reference in its entirety) have constructed a Markov model describing this process. In certain embodiments of the invention, this model may be used to determine the composition of the N1 and/or N2 segments used in libraries of the invention. Alternatively, the parameters presented in Jackson et al. could be further refined to produce sequences that more closely mimic human sequences.

2.2.4. Design of a CDRH3 Library Using the N1, DH, N2, and H3-JH Segments

The CDRH3 libraries of the invention comprise an initial amino acid (in certain exemplary embodiments, G, D, E) or lack thereof (designated herein as position 95), followed by the N1, DH, N2, and H3-JH segments. Thus, in certain embodiments of the invention, the overall design of the CDRH3 libraries can be represented by the following formula:

[G/D/E/-]-[N1]-[DH]-[N2]-[H3-JH].

While the compositions of each portion of a CDRH3 of a library of the invention are more fully described above, the composition of the tail presented above (G/D/E/-) is non-limiting, and that any amino acid (or no amino acid) can be used in this position. Thus, certain embodiments of the invention may be represented by the following formula:

[X]-[N1]-[DH]-[N2]-[H3-JH],

wherein [X] is any amino acid residue or no residue.

In certain embodiments of the invention, a synthetic CDRH3 repertoire is combined with selected VH chassis sequences and heavy chain constant regions, via homologous recombination. Therefore, in certain embodiments of the invention, it may be necessary to include DNA sequences flanking the 5′ and 3′ ends of the synthetic CDRH3 libraries, to facilitate homologous recombination between the synthetic CDRH3 libraries and vectors containing the selected chassis and constant regions. In certain embodiments, the vectors also contain a sequence encoding at least a portion of the non-nibbled region of the IGHJ gene (i.e., FRM4-JH). Thus, a polynucleotide encoding an N-terminal sequence (e.g. CA(K/R/T)) may be added to the synthetic CDRH3 sequences, wherein the N-terminal polynucleotide is homologous with FRM3 of the chassis, while a polynucleotide encoding a C-terminal sequence (e.g., WG(Q/R)G; SEQ ID NO: 23) may be added to the synthetic CDRH3, wherein the C-terminal polynucleotide is homologous with FRM4-JH. Although the sequence WG(Q/R)G (SEQ ID NO: 23) is presented in this exemplary embodiment, additional amino acids, C-terminal to this sequence in FRM4-JH may also be included in the polynucleotide encoding the C-terminal sequence. The purpose of the polynucleotides encoding the N-terminal and C-terminal sequences, in this case, is to facilitate homologous recombination, and one of ordinary skill in the art would recognize that these sequences may be longer or shorter than depicted below. Accordingly, in certain embodiments of the invention, the overall design of the CDRH3 repertoire, including the sequences required to facilitate homologous recombination with the selected chassis, can be represented by the following formula (regions homologous with vector underlined):

CA[R/K/T]-[X]-[N1]-[DH]-[N2]-[H3-JH]-[WG(Q/R)G].

In other embodiments of the invention, the CDRH3 repertoire can be represented by the following formula, which excludes the T residue presented in the schematic above:

CA[R/K]-[X]-[N1]-[DH]-[N2]-[H3-JH]-[WG(Q/R)G].

References describing collections of V, D, and J genes include Scaviner et al., Exp. Clin, Immunogenet., 1999, 16: 243 and Ruiz et al., Exp. Clin. Immunogenet, 1999, 16: 173, each incorporated by reference in its entirety.

2.2.5. CDRH3 Length Distributions

As described throughout this application, in addition to accounting for the composition of naturally occurring CDRH3 segments, the instant invention also takes into account the length distribution of naturally occurring CDRH3 segments. Surveys by Zemlin et al. (JMB, 2003, 334: 733, incorporated by reference in its entirety) and Lee et al. (Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety) provide analyses of the naturally occurring CDRH3 lengths. These data show that about 95% of naturally occurring CDRH3 sequences have a length from about 7 to about 23 amino acids. In certain embodiments, the instant invention provides rationally designed antibody libraries with CDRH3 segments which directly mimic the size distribution of naturally occurring CDRH3 sequences. In certain embodiments of the invention, the length of the CDRH3s may be about 2 to about 30, about 3 to about 35, about 7 to about 23, about 3 to about 28, about 5 to about 28, about 5 to about 26, about 5 to about 24, about 7 to about 24, about 7 to about 22, about 8 to about 19, about 9 to about 22, about 9 to about 20, about 10 to about 18, about 11 to about 20, about 11 to about 18, about 13 to about 18, or about 13 to about 16 residues in length.

In certain embodiments of the invention, the length distribution of a CDRH3 library of the invention may be defined based on the percentage of sequences within a certain length range. For example, in certain embodiments of the invention, CDRH3s with a length of about 10 to about 18 amino acid residues comprise about 84% to about 94% of the sequences of a the library. In some embodiments, sequences within this length range comprise about 89% of the sequences of a library.

In other embodiments of the invention, CDRH3s with a length of about 11 to about 17 amino acid residues comprise about 74% to about 84% of the sequences of a library. In some embodiments, sequences within this length range comprise about 79% of the sequences of a library.

In still other embodiments of the invention, CDRH3s with a length of about 12 to about 16 residues comprise about 57% to about 67% of the sequences of a library. In some embodiments, sequences within this length range comprise about 62% of the sequences of a library.

In certain embodiments of the invention, CDRH3s with a length of about 13 to about 15 residues comprise about 35% to about 45% of the sequences of a library. In some embodiments, sequences within this length range comprise about 40% of the sequences of a library.

2.3. Design of the Antibody Library CDRL3 Components

The CDRL3 libraries of the invention can be generated by one of several approaches. The actual version of the CDRL3 library made and used in a particular embodiment of the invention will depend on objectives for the use of the library. More than one CDRL3 library may be used in a particular embodiment; for example, a library containing CDRH3 diversity, with kappa and lambda light chains is within the scope of the invention.

In certain embodiments of the invention, a CDRL3 library is a VKCDR3 (kappa) library and/or a VλCDR3 (lambda) library. The CDRL3 libraries described herein differ significantly from CDRL3 libraries in the art. First, they consider length variation that is consistent with what is observed in actual human sequences. Second, they take into consideration the fact that a significant portion of the CDRL3 is encoded by the IGLV gene. Third, the patterns of amino acid variation within the IGLV gene-encoded CDRL3 portions are not stochastic and are selected based on depending on the identity of the IGLV gene. Taken together, the second and third distinctions mean that CDRL3 libraries that faithfully mimic observed patterns in human sequences cannot use a generic design that is independent of the chassis sequences in FRM1 to FRM3. Fourth, the contribution of JL to CDRL3 is also considered explicitly, and enumeration of each amino acid residue at the relevant positions is based on the compositions and natural variations of the JL genes themselves.

As indicated above, and throughout the application, a unique aspect of the design of the libraries of the invention is the germline or “chassis-based” aspect, which is meant to preserve more of the integrity and variability of actual human sequences. This is in contrast to other codon-based synthesis or degenerate oligonucleotide synthesis approaches that have been described in the literature and that aim to produce “one-size-fits-all” (e.g., consensus) libraries (e.g., Knappik, et al., J Mol Biol, 2000, 296: 57; Akamatsu et al., J Immunol, 1993, 151: 4651, each incorporated by reference in its entirety).

In certain embodiments of the invention, patterns of occurrence of particular amino acids at defined positions within VL sequences are determined by analyzing data available in public or other databases, for example, the NCBI database (see, for example, GI numbers in Appendices A and B filed herewith). In certain embodiments of the invention, these sequences are compared on the basis of identity and assigned to families on the basis of the germline genes from which they are derived. The amino acid composition at each position of the sequence, in each germline family, may then be determined. This process is illustrated in the Examples provided herein.

2.3.1. Minimalist VKCDR3 Libraries

In certain embodiments of the invention, the light chain CDR3 library is a VKCDR3 library. Certain embodiments of the invention may use only the most common VKCDR3 length, nine residues; this length occurs in a dominant proportion (greater than about 70%) of human VKCDR3 sequences. In human VKCDR3 sequences of length nine, positions 89-95 are encoded by the IGKV gene and positions 96-97 are encoded by the IGKJ gene. Analysis of human kappa light chain sequences indicates that there are not strong biases in the usage of the IGKJ genes. Therefore, in certain embodiments of the invention, each of the five the IGKJ genes can be represented in equal proportions to create a combinatorial library of (M VK chassis)×(5 JK genes), or a library of size M×5. However, in other embodiments of the invention, it may be desirable to bias IGKJ gene representation, for example to restrict the size of the library or to weight the library toward IGKJ genes known to have particular properties.

As described in Example 6.1, examination of the first amino acid encoded by the IGKJ gene (position 96) indicated that the seven most common residues found at this position are L, Y, R, W, F, P, and I. These residues cumulatively account for about 85% of the residues found in position 96 in naturally occurring kappa light chain sequences. In certain embodiments of the invention, the amino acid residue at position 96 may be one of these seven residues. In other embodiments of the invention, the amino acid at this position may be chosen from amongst any of the other 13 amino acid residues. In still other embodiments of the invention, the amino acid residue at position 96 may be chosen from amongst the top 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids that occur at position 96, or even residues that never occur at position 96. Similarly, the occurrence of the amino acids selected to occupy position 96 may be equivalent or weighted. In certain embodiments of the invention, it may be desirable to include each of the amino acids selected for inclusion in position 96 at equivalent amounts. In other embodiments of the invention, it may be desirable to bias the composition of position 96 to include particular residues more or less frequently than others. For example, as presented in Example 6.1, arginine occurs at position 96 most frequently when the IGKJ1 germline sequence is used. Therefore, in certain embodiments of the invention, it may be desirable to bias amino acid usage at position 96 according to the origin of the IGKJ germline sequence(s) and/or the IGKV germline sequence(s) selected for representation in a library.

Therefore, in certain embodiments of the invention, a minimalist VKCDR3 library may be represented by one or more of the following amino acid sequences:

[VK_Chassis]-[L3-VK]-[F/L/I/R/W/Y/P]-[JK*]

[VK_Chassis]-[L3-VK]-[X]-[JK*]

In these schematic exemplary sequences, VK_Chassis represents any VK chassis selected for inclusion in a library of the invention (e.g., see Table 11). Specifically, VK_Chassis comprises about Kabat residues 1 to 88 of a selected IGKV sequence. L3-VK represents the portion of the VKCDR3 encoded by the chosen IGKV gene (in this embodiment, Kabat residues 89-95). F, L, I, R, W, Y, and P are the seven most commonly occurring amino acids at position 96 of VKCDR3s with length nine, X is any amino acid, and JK* is an IGKJ amino acid sequence without the N-terminal residue (i.e., the N-terminal residue is substituted with F, L, I, R, W, Y, P, or X). Thus, in one possible embodiment of the minimalist VKCDR3 library, 70 members could be produced by utilizing 10 VK chassis, each paired with its respective L3-VK, 7 amino acids at position 96 (i.e., X), and one JK* sequence. Another embodiment of the library may have 350 members, produced by combining 10 VK chassis, each paired with its respective L3-VK, with 7 amino acids at position 96, and all 5 JK* genes. Still another embodiment of the library may have 1,125 members, produced by combining 15 VK chassis, each paired with its respective H3-JK, with 15 amino acids at position 96 and all 5 JK* genes, and so on. A person of ordinary skill in the art will readily recognize that many other combinations are possible. Moreover, while it is believed that maintaining the pairing between the VK chassis and the L3-VK results in libraries that are more similar to human kappa light chain sequences in composition, the L3-VK regions may also be combinatorially varied with different VK chassis regions, to create additional diversity.

2.3.2. VKCDR3 Libraries of About 10⁵ Complexity

While the dominant length of VKCDR3 sequences in humans is about nine amino acids, other lengths appear at measurable frequencies that cumulatively approach almost about 30% of VKCDR3 sequences. In particular, VKCDR3 of lengths 8 and 10 represent about 8.5% and about 16%, respectively, of VKCDR3 lengths in representative samples (Example 6.2; FIG. 3). Thus, more complex VKCDR3 libraries may include CDR lengths of 8, 10, and 11 amino acids. Such libraries could account for a greater percentage of the length distribution observed in collections of human VKCDR3 sequences, or even introduce VKCDR3 lengths that do not occur frequently in human VKCDR3 sequences (e.g., less than eight residues or greater than 11 residues).

The inclusion of a diversity of kappa light chain length variations in a library of the invention also enables one to include sequence variability that occurs outside of the amino acid at the VK-JK junction (i.e., position 96, described above). In certain embodiments of the invention, the patterns of sequence variation within the VK, and/or JK segments can be determined by aligning collections of sequences derived from particular germline sequences. In certain embodiments of the invention, the frequency of occurrence of amino acid residues within VKCDR3 can be determined by sequence alignments (e.g., see Example 6.2 and Table 30). In some embodiments of the invention, this frequency of occurrence may be used to introduce variability into the VK_Chassis, L3-VK and/or JK segments that are used to synthesize the VKCDR3 libraries. In certain embodiments of the invention, the top 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids that occur at any particular position in a naturally occurring repertoire may be included at that position in a VKCDR3 library of the invention. In certain embodiments of the invention, the percent occurrence of any amino acid at any particular position within the VKCDR3 or a VK light chain may be about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments of the invention, the percent occurrence of any amino acid at any position within a VKCDR3 or kappa light chain library of the invention may be within at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, or 200% of the percent occurrence of any amino acid at any position within a naturally occurring VKCDR3 or kappa light chain domain.

In some embodiments of the invention, a VKCDR3 library may be synthesized using degenerate oligonucleotides (see Table 31 for IUPAC base symbol definitions). In some embodiments of the invention, the limits of oligonucleotide synthesis and the genetic code may require the inclusion of more or fewer amino acids at a particular position in the VKCDR3 sequences. An illustrative embodiment of this approach is provided in Example 6.2.

2.3.3. More Complex VKCDR3 Libraries

The limitations inherent in using the genetic code and degenerate oligonucleotide synthesis may, in some cases, require the inclusion of more or fewer amino acids at a particular position within VKCDR3 (e.g., Example 6.2, Table 32), in comparison to those amino acids found at that position in nature. This limitation can be overcome through the use of a codon-based synthesis approach (Virnekas et al. Nucleic Acids Res., 1994, 22: 5600, incorporated by reference in its entirety), which enables precise synthesis of oligonucleotides encoding particular amino acids and a finer degree of control over the proportion of any particular amino acid incorporated at any position. Example 6.3 describes this approach in greater detail.

In some embodiments of the invention, a codon-based synthesis approach may be used to vary the percent occurrence of any amino acid at any particular position within the VKCDR3 or kappa light chain. In certain embodiments, the percent occurrence of any amino acid at any position in a VKCDR3 or kappa light chain sequence of the library may be about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments of the invention, the percent occurrence of any amino acid at any position may be about 1%, 2%, 3%, or 4%. In certain embodiments of the invention, the percent occurrence of any amino acid at any position within a VKCDR3 or kappa light chain library of the invention may be within at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, or 200% of the percent occurrence of any amino acid at any position within a naturally occurring VKCDR3 or kappa light chain domain.

In certain embodiments of the invention, the VKCDR3 (and any other sequence used in the library, regardless of whether or not it is part of VKCDR3) may be altered to remove undesirable amino acid motifs. For example, peptide sequences with the pattern N-X-(S or T)-Z, where X and Z are different from P, will undergo post-translational modification (N-linked glycosylation) in a number of expression systems, including yeast and mammalian cells. In certain embodiments of the invention, the introduction of N residues at certain positions may be avoided, so as to avoid the introduction of N-linked glycosylation sites. In some embodiments of the invention, these modifications may not be necessary, depending on the organism used to express the library and the culture conditions. However, even in the event that the organism used to express libraries with potential N-linked glycosylation sites is incapable of N-linked glycosylation (e.g., bacteria), it may still be desirable to avoid N-X-(S/T) sequences, as the antibodies isolated from such libraries may be expressed in different systems (e.g., yeast, mammalian cells) later (e.g., toward clinical development), and the presence of carbohydrate moieties in the variable domains, and the CDRs in particular, may lead to unwanted modifications of activity.

In certain embodiments of the invention, it may be preferable to create the individual sub-libraries of different lengths (e.g., one or more of lengths 5, 6, 7, 8, 9, 10, 11, or more) separately, and then mix the sub-libraries in proportions that reflect the length distribution of VKCDR3 in human sequences; for example, in ratios approximating the 1:9:2 distribution that occurs in natural VKCDR3 sequences of lengths 8, 9, and 10 (see FIG. 3). In other embodiments, it may be desirable to mix these sub-libraries at ratios that are different from the distribution of lengths in natural VKCDR3 sequences, for example, to produce more focused libraries or libraries with particular properties.

2.3.4. VλCDR3 Libraries

The principles used to design the minimalist VλCDR3 libraries of the invention are similar to those enumerated above, for the VKCDR3 libraries, and are explained in more detail in the Examples. One difference between the VλCDR3 libraries of the invention and the VKCDR3 libraries of the invention is that, unlike the IGKV genes, the contribution of the IGVλ genes to CDRL3 (i.e., L3-Vλ) is not constrained to a fixed number of amino acid residues. Therefore, while the combination of the VK (including L3-VK) and JK segments, with inclusion of position 96, yields CDRL3 with a length of only 9 residues, length variation may be obtained within a VλCDR3 library even when only the Vλ (including L3-Vλ) and Jλ segments are considered.

As for the VKCDR3 sequences, additional variability may be introduced into the VλCDR3 sequences via the same methods outlined above, namely determining the frequency of occurrence of particular residues within VλCDR3 sequences and synthesizing the oligonucleotides encoding the desired compositions via degenerate oligonucleotide synthesis or trinucleotides-based synthesis.

2.4. Synthetic Antibody Libraries

In certain embodiments of the invention, both the heavy and light chain chassis sequences and the heavy and light chain CDR3 sequences are synthetic. The polynucleotide sequences of the instant invention can be synthesized by various methods. For example, sequences can be synthesized by split pool DNA synthesis as described in Feldhaus et al., Nucleic Acids Research, 2000, 28: 534; Omstein et al., Biopolymers, 1978, 17: 2341; and Brenner and Lerner, PNAS, 1992, 87: 6378 (each of which is incorporated by reference in its entirety).

In some embodiments of the invention, cassettes representing the possible V, D, and J diversity found in the human repertoire, as well as junctional diversity, are synthesized de novo either as double-stranded DNA oligonucleotides, single-stranded DNA oligonucleotides representative of the coding strand, or single-stranded DNA oligonucleotides representative of the non-coding strand. These sequences can then be introduced into a host cell along with an acceptor vector containing a chassis sequence and, in some cases a portion of FRM4 and a constant region. No primer-based PCR amplification from mammalian cDNA or mRNA or template-directed cloning steps from mammalian cDNA or mRNA need be employed.

2.5. Construction of Libraries by Yeast Homologous Recombination

In certain embodiments, the present invention exploits the inherent ability of yeast cells to facilitate homologous recombination at high efficiency. The mechanism of homologous recombination in yeast and its applications are briefly described below.

As an illustrative embodiment, homologous recombination can be carried out in, for example, Saccharomyces cerevisiae, which has genetic machinery designed to carry out homologous recombination with high efficiency. Exemplary S. cerevisiae strains include EM93, CEN.PK2, RM11-1a, YJM789, and BJ5465. This mechanism is believed to have evolved for the purpose of chromosomal repair, and is also called “gap repair” or “gap filling”. By exploiting this mechanism, mutations can be introduced into specific loci of the yeast genome. For example, a vector carrying a mutant gene can contain two sequence segments that are homologous to the 5′ and 3′ open reading frame (ORF) sequences of a gene that is intended to be interrupted or mutated. The vector may also encode a positive selection marker, such as a nutritional enzyme allele (e.g. URA3) and/or an antibiotic resistant marker (e.g., Geneticin/G418), flanked by the two homologous DNA segments. Other selection markers and antibiotic resistance markers are known to one of ordinary skill in the art. In some embodiments of the invention, this vector (e.g., a plasmid) is linearized and transformed into the yeast cells. Through homologous recombination between the plasmid and the yeast genome, at the two homologous recombination sites, a reciprocal exchange of the DNA content occurs between the wild type gene in the yeast genome and the mutant gene (including the selection marker gene(s)) that is flanked by the two homologous sequence segments. By selecting for the one or more selection markers, the surviving yeast cells will be those cells in which the wild-type gene has been replaced by the mutant gene (Pearson et al., Yeast, 1998, 14: 391, incorporated by reference in its entirety). This mechanism has been used to make systematic mutations in all 6,000 yeast genes, or open reading frames (ORFs), for functional genomics studies. Because the exchange is reciprocal, a similar approach has also been used successfully to clone yeast genomic DNA fragments into a plasmid vector (Iwasaki et al., Gene, 1991, 109: 81, incorporated by reference in its entirety).

By utilizing the endogenous homologous recombination machinery present in yeast, gene fragments or synthetic oligonucleotides can also be cloned into a plasmid vector without a ligation step. In this application of homologous recombination, a target gene fragment (i.e., the fragment to be inserted into a plasmid vector, e.g., a CDR3) is obtained (e.g., by oligonucleotides synthesis, PCR amplification, restriction digestion out of another vector, etc.). DNA sequences that are homologous to selected regions of the plasmid vector are added to the 5′ and 3′ ends of the target gene fragment. These homologous regions may be fully synthetic, or added via PCR amplification of a target gene fragment with primers that incorporate the homologous sequences. The plasmid vector may include a positive selection marker, such as a nutritional enzyme allele (e.g., URA3), or an antibiotic resistance marker (e.g., Geneticin/G418). The plasmid vector is then linearized by a unique restriction cut located in-between the regions of sequence homology shared with the target gene fragment, thereby creating an artificial gap at the cleavage site. The linearized plasmid vector and the target gene fragment flanked by sequences homologous to the plasmid vector are co-transformed into a yeast host strain. The yeast is then able to recognize the two stretches of sequence homology between the vector and target gene fragment and facilitate a reciprocal exchange of DNA content through homologous recombination at the gap. As a consequence, the target gene fragment is inserted into the vector without ligation.

The method described above has also been demonstrated to work when the target gene fragments are in the form of single stranded DNA, for example, as a circular M13 phage derived form, or as single stranded oligonucleotides (Simon and Moore, Mol. Cell. Biol., 1987, 7: 2329; Ivanov et al., Genetics, 1996, 142: 693; and DeMarini et al., 2001, 30: 520., each incorporated by reference in its entirety). Thus, the form of the target that can be recombined into the gapped vector can be double stranded or single stranded, and derived from chemical synthesis, PCR, restriction digestion, or other methods.

Several factors may influence the efficiency of homologous recombination in yeast. For example, the efficiency of the gap repair is correlated with the length of the homologous sequences flanking both the linearized vector and the target gene. In certain embodiments, about 20 or more base pairs may be used for the length of the homologous sequence, and about 80 base pairs may give a near-optimized result (Hua et al., Plasmid, 1997, 38: 91; Raymond et al., Genome Res., 2002, 12: 190, each incorporated by reference in its entirety). In certain embodiments of the invention, at least about 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 187, 190, or 200 homologous base pairs may be used to facilitate recombination. In other embodiments, between about 20 and about 40 base pairs are utilized. In addition, the reciprocal exchange between the vector and gene fragment is strictly sequence-dependent, i.e. it does not cause a frame shift. Therefore, gap-repair cloning assures the insertion of gene fragments with both high efficiency and precision. The high efficiency makes it possible to clone two, three, or more targeted gene fragments simultaneously into the same vector in one transformation attempt (Raymond et al., Biotechniques, 1999, 26: 134, incorporated by reference in its entirety). Moreover, the nature of precision sequence conservation through homologous recombination makes it possible to clone selected genes or gene fragments into expression or fusion vectors for direct functional examination (El-Deiry et al., Nature Genetics, 1992, 1: 4549; Ishioka et al., PNAS, 1997, 94: 2449, each incorporated by reference in its entirety).

Libraries of gene fragments have also been constructed in yeast using homologous recombination. For example, a human brain cDNA library was constructed as a two-hybrid fusion library in vector pJG4-5 (Guidotti and Zervos, Yeast, 1999, 15: 715, incorporated by reference in its entirety). It has also been reported that a total of 6,000 pairs of PCR primers were used for amplification of 6,000 known yeast ORFs for a study of yeast genomic protein interactions (Hudson et al., Genome Res., 1997, 7: 1169, incorporated by reference in its entirety). In 2000, Uetz et al. conducted a comprehensive analysis-of protein-protein interactions in Saccharomyces cerevisiae (Uetz et al., Nature, 2000, 403: 623, incorporated by reference in its entirety). The protein-protein interaction map of the budding yeast was studied by using a comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins (Ito et al., PNAS, 2000, 97: 1143, incorporated by reference in its entirety), and the genomic protein linkage map of Vaccinia virus was studied using this system (McCraith et al., PNAS, 2000, 97: 4879, incorporated by reference in its entirety).

In certain embodiments of the invention, a synthetic CDR3 (heavy or light chain) may be joined by homologous recombination with a vector encoding a heavy or light chain chassis, a portion of FRM4, and a constant region, to form a full-length heavy or light chain. In certain embodiments of the invention, the homologous recombination is performed directly in yeast cells. In some embodiments, the method comprises:

-   -   (a) transforming into yeast cells:         -   (i) a linearized vector encoding a heavy or light chain             chassis, a portion of FRM4, and a constant region, wherein             the site of linearization is between the end of FRM3 of the             chassis and the beginning of the constant region; and         -   (ii) a library of CDR3 insert nucleotide sequences that are             linear and double stranded, wherein each of the CDR3 insert             sequences comprises a nucleotide sequence encoding CDR3 and             5′- and 3′-flanking sequences that are sufficiently             homologous to the termini of the vector of (i) at the site             of linearization to enable homologous recombination to occur             between the vector and the library of CDR3 insert sequences;             and     -   (b) allowing homologous recombination to occur between the         vector and the CDR3 insert sequences in the transformed yeast         cells, such that the CDR3 insert sequences are incorporated into         the vector, to produce a vector encoding full-length heavy chain         or light chain.

As specified above, the CDR3 inserts may have a 5′ flanking sequence and a 3′ flanking sequence that are homologous to the termini of the linearized vector. When the CDR3 inserts and the linearized vectors are introduced into a host cell, for example, a yeast cell, the “gap” (the linearization site) created by linearization of the vector is filled by the CDR3 fragment insert through recombination of the homologous sequences at the 5′ and 3′ termini of these two linear double-stranded DNAs (i.e., the vector and the insert). Through this event of homologous recombination, libraries of circular vectors encoding full-length heavy or light chains comprising variable CDR3 inserts is generated. Particular instances of these methods are presented in the Examples.

Subsequent analysis may be carried out to determine the efficiency of homologous recombination that results in correct insertion of the CDR3 sequences into the vectors. For example, PCR amplification of the CDR3 inserts directly from selected yeast clones may reveal how many clones are recombinant. In certain embodiments, libraries with minimum of about 90% recombinant clones are utilized. In certain other embodiments libraries with a minimum of about 1%, 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% recombinant clones are utilized. The same PCR amplification of selected clones may also reveal the insert size.

To verify the sequence diversity of the inserts in the selected clones, a PCR amplification product with the correct size of insert may be “fingerprinted” with restriction enzymes known to cut or not cut within the amplified region. From a gel electrophoresis pattern, it may be determined whether the clones analyzed are of the same identity or of the distinct or diversified identity. The PCR products may also be sequenced directly to reveal the identity of inserts and the fidelity of the cloning procedure, and to prove the independence and diversity of the clones. FIG. 1 depicts a schematic of recombination between a fragment (e.g., CDR3) and a vector (e.g. comprising a chassis, portion of FRM4, and constant region) for the construction of a library.

2.6. Expression and Screening Systems

Libraries of polynucleotides generated by any of the techniques described herein, or other suitable techniques, can be expressed and screened to identify antibodies having desired structure and/or activity. Expression of the antibodies can be carried out, for example, using cell-free extracts (and e.g., ribosome display), phage display, prokaryotic cells (e.g., bacterial display), or eukaryotic cells (e.g. yeast display). In certain embodiments of the invention, the antibody libraries are expressed in yeast.

In other embodiments, the polynucleotides are engineered to serve as templates that can be expressed in a cell-free extract. Vectors and extracts as described, for example in U.S. Pat. Nos. 5,324,637; 5,492,817; 5,665,563, (each incorporated by reference in its entirety) can be used and many are commercially available. Ribosome display and other cell-free techniques for linking a polynucleotide (i.e., a genotype) to a polypeptide (i.e., a phenotype) can be used, e.g., Profusion™ (see, e.g. U.S. Pat. Nos. 6,348,315; 6,261,804; 6,258,558; and 6,214,553, each incorporated by reference in its entirety).

Alternatively, the polynucleotides of the invention can be expressed in an E. coli expression system, such as that described by Pluckthun and Skerra. (Meth. Enzymol., 1989, 178: 476; Biotechnology, 1991, 9: 273, each incorporated by reference in its entirety). The mutant proteins can be expressed for secretion in the medium and/or in the cytoplasm of the bacteria, as described by Better and Horwitz, Meth. Enzymol., 1989, 178: 476, incorporated by reference in its entirety. In some embodiments, the single domains encoding VH and VL are each attached to the 3′ end of a sequence encoding a signal sequence, such as the ompA, phoA or pelB signal sequence (Lei et al., J. Bacteriol., 1987, 169: 4379, incorporated by reference in its entirety). These gene fusions are assembled in a dicistronic construct, so that they can be expressed from a single vector, and secreted into the periplasmic space of E. coli where they will refold and can be recovered in active form. (Skerra et al., Biotechnology, 1991, 9: 273, incorporated by reference in its entirety). For example, antibody heavy chain genes can be concurrently expressed with antibody light chain genes to produce antibodies or antibody fragments.

In other embodiments of the invention, the antibody sequences are expressed on the membrane surface of a prokaryote, e.g., E. coli, using a secretion signal and lipidation moiety as described, e.g., in US20040072740; US20030100023; and US20030036092 (each incorporated by reference in its entirety).

Higher eukaryotic cells, such as mammalian cells, for example myeloma cells (e.g., NS/0 cells), hybridoma cells, Chinese hamster ovary (CHO), and human embryonic kidney (HEK) cells, can also be used for expression of the antibodies of the invention. Typically, antibodies expressed in mammalian cells are designed to be secreted into the culture medium, or expressed on the surface of the cell. The antibody or antibody fragments can be produced, for example, as intact antibody molecules or as individual VH and VL fragments, Fab fragments, single domains, or as single chains (scFv) (Huston et al., PNAS, 1988, 85: 5879, incorporated by reference in its entirety).

Alternatively, antibodies can be expressed and screened by anchored periplasmic expression (APEx 2-hybrid surface display), as described, for example, in Jeong et al., PNAS, 2007, 104: 8247 (incorporated by reference in its entirety) or by other anchoring methods as described, for example, in Mazor et al., Nature Biotechnology, 2007, 25: 563 (incorporated by reference in its entirety).

In other embodiments of the invention, antibodies can be selected using mammalian cell display (Ho et al., PNAS, 2006, 103: 9637, incorporated by reference in its entirety).

The screening of the antibodies derived from the libraries of the invention can be carried out by any appropriate means. For example, binding activity can be evaluated by standard immunoassay and/or affinity chromatography. Screening of the antibodies of the invention for catalytic function, e.g., proteolytic function can be accomplished using a standard assays, e.g. the hemoglobin plaque assay as described in U.S. Pat. No. 5,798,208 (incorporated by reference in its entirety). Determining the ability of candidate antibodies to bind therapeutic targets can be assayed in vitro using, e.g. a BIACORE™ instrument, which measures binding rates of an antibody to a given target or antigen based on surface plasmon resonance. In vivo assays can be conducted using any of a number of animal models and then subsequently tested, as appropriate, in humans. Cell-based biological assays are also contemplated.

One aspect of the instant invention is the speed at which the antibodies of the library can be expressed and screened. In certain embodiments of the invention, the antibody library can be expressed in yeast, which have a doubling time of less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the doubling times are about 1 to about 3 hours, about 2 to about 4, about 3 to about 8 hours, about 3 to about 24, about 5 to about 24, about 4 to about 6 about 5 to about 22, about 6 to about 8, about 7 to about 22, about 8 to about 10 hours, about 7 to about 20, about 9 to about 20, about 9 to about 18, about 11 to about 18, about 11 to about 16, about 13 to about 16, about 16 to about 20, or about 20 to about 30 hours. In certain embodiments of the invention, the antibody library is expressed in yeast with a doubling time of about 16 to about 20 hours, about 8 to about 16 hours, or about 4 to about 8 hours. Thus, the antibody library of the instant invention can be expressed and screened in a matter of hours, as compared to previously known techniques which take several days to express and screen antibody libraries. A limiting step in the throughput of such screening processes in mammalian cells is simply the time required to iteratively regrow populations of isolated cells, which, in some cases, have doubling times greater than the doubling times of the yeast used in the current invention.

In certain embodiments of the invention, the composition of a library may be defined after one or more enrichment steps (for example by screening for antigen binding, or other properties). For example, a library with a composition comprising about x % sequences or libraries of the invention may be enriched to contain about 2x %, 3x %, 4x %, 5x %, 6x %, 7x %, 8x %, 9x %, 10x %, 20x %, 25x %, 40x %, 50x %, 60x % 75x %, 80x %, 90x %, 95x %, or 99x % sequences or libraries of the invention, after one or more screening steps. In other embodiments of the invention, the sequences or libraries of the invention may be enriched about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1,000-fold, or more, relative to their occurrence prior to the one or more enrichment steps. In certain embodiments of the invention, a library may contain at least a certain number of a particular type of sequence(s), such as CDRH3s, CDRL3s, heavy chains, light chains, or whole antibodies (e.g. at least about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, or 10²⁰). In certain embodiments, these sequences may be enriched during one or more enrichment steps, to provide libraries comprising at least about 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, or 10¹⁹ of the respective sequence(s).

2.7. Mutagenesis Approaches for Affinity Maturation

As described above, antibody leads can be identified through a selection process that involves screening the antibodies of a library of the invention for binding to one or more antigens, or for a biological activity. The coding sequences of these antibody leads may be further mutagenized in vitro or in vivo to generate secondary libraries with diversity introduced in the context of the initial antibody leads. The mutagenized antibody leads can then be further screened for binding to target antigens or biological activity, in vitro or in vivo, following procedures similar to those used for the selection of the initial antibody lead from the primary library. Such mutagenesis and selection of primary antibody leads effectively mimics the affinity maturation process naturally occurring in a mammal that produces antibodies with progressive increases in the affinity to an antigen. In one embodiment of the invention, only the CDRH3 region is mutagenized. In another embodiment of the invention, the whole variable region is mutagenized. In other embodiments of the invention one or more of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/ CDRL3 may be mutagenized. In some embodiments of the invention, “light chain shuffling” may be used as part of the affinity maturation protocol. In certain embodiments, this may involve pairing one or more heavy chains with a number of light chains, to select light chains that enhance the affinity and/or biological activity of an antibody. In certain embodiments of the invention, the number of light chains to which the one or more heavy chains can be paired is at least about 2, 5, 10, 100, 1000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰. In certain embodiments of the invention, these light chains are encoded by plasmids. In other embodiments of the invention, the light chains may be integrated into the genome of the host cell.

The coding sequences of the antibody leads may be mutagenized by a wide variety of methods. Examples of methods of mutagenesis include, but are not limited to site-directed mutagenesis, error-prone PCR mutagenesis, cassette mutagenesis, and random PCR mutagenesis. Alternatively, oligonucleotides encoding regions with the desired mutations can be synthesized and introduced into the sequence to be mutagenized, for example, via recombination or ligation.

Site-directed mutagenesis or point mutagenesis may be used to gradually change the CDR sequences in specific regions. This may be accomplished by using oligonucleotide-directed mutagenesis or PCR. For example, a short sequence of an antibody lead may be replaced with a synthetically mutagenized oligonucleotide in either the heavy chain or light chain region, or both. The method may not be efficient for mutagenizing large numbers of CDR sequences, but may be used for fine tuning of a particular lead to achieve higher affinity toward a specific target protein.

Cassette mutagenesis may also be used to mutagenize the CDR sequences in specific regions. In a typical cassette mutagenesis, a sequence block, or a region, of a single template is replaced by a completely or partially randomized sequence. However, the maximum information content that can be obtained may be statistically limited by the number of random sequences of the oligonucleotides. Similar to point mutagenesis, this method may also be used for fine tuning of a particular lead to achieve higher affinity towards a specific target protein.

Error-prone PCR, or “poison” PCR, may be used to mutagenize the CDR sequences by following protocols described in Caldwell and Joyce, PCR Methods and Applications, 1992, 2: 28; Leung et al., Technique, 1989, 1: 11; Shafikhani et al., Biotechniques, 1997, 23: 304; and Stemmer et al., PNAS, 1994, 91: 10747 (each of which is incorporated by reference in its entirety).

Conditions for error prone PCR may include (a) high concentrations of Mn²⁺ (e.g., about 0.4 to about 0.6 mM) that efficiently induces malfunction of Taq DNA polymerase; and (b) a disproportionally high concentration of one nucleotide substrate (e.g., dGTP) in the PCR reaction that causes incorrect incorporation of this high concentration substrate into the template and produces mutations. Additionally, other factors such as, the number of PCR cycles, the species of DNA polymerase used, and the length of the template, may affect the rate of misincorporation of “wrong” nucleotides into the PCR product. Commercially available kits may be utilized for the mutagenesis of the selected antibody library, such as the “Diversity PCR random mutagenesis kit” (CLONTECH™).

The primer pairs used in PCR-based mutagenesis may, in certain embodiments, include regions matched with the homologous recombination sites in the expression vectors. This design allows facile re-introduction of the PCR products back into the heavy or light chain chassis vectors, after mutagenesis, via homologous recombination.

Other PCR-based mutagenesis methods can also be used, alone or in conjunction with the error prone PCR described above. For example, the PCR amplified CDR segments may be digested with DNase to create nicks in the double stranded DNA. These nicks can be expanded into gaps by other exonucleases such as Bal 31. The gaps may then be filled by random sequences by using DNA Klenow polymerase at a low concentration of regular substrates dGTP, dATP, dTTP, and dCTP with one substrate (e.g., dGTP) at a disproportionately high concentration. This fill-in reaction should produce high frequency mutations in the filled gap regions. These method of DNase digestion may be used in conjunction with error prone PCR to create a high frequency of mutations in the desired CDR segments.

The CDR or antibody segments amplified from the primary antibody leads may also be mutagenized in vivo by exploiting the inherent ability of mutation in pre-B cells. The Ig genes in pre-B cells are specifically susceptible to a high-rate of mutation. The Ig promoter and enhancer facilitate such high rate mutations in a pre-B cell environment while the pre-B cells proliferate. Accordingly, CDR gene segments may be cloned into a mammalian expression vector that contains a human Ig enhancer and promoter. This construct may be introduced into a pre-B cell line, such as 38B9, which allows the mutation of the VH and VL gene segments naturally in the pre-B cells (Liu and Van Ness, Mol. Immunol., 1999, 36: 461, incorporated by reference in its entirety). The mutagenized CDR segments can be amplified from the cultured pre-B cell line and re-introduced back into the chassis-containing vector(s) via, for example, homologous recombination.

In some embodiments, a CDR “hit” isolated from screening the library can be re-synthesized, using degenerate codons or trinucleotides, and re-cloned into the heavy or light chain vector using gap repair.

3. Library Sampling

In certain embodiments of the invention, a library of the invention comprises a designed, non-random repertoire wherein the theoretical diversity of particular components of the library (for example, CDRH3), but not necessarily all components or the entire library, can be over-sampled in a physical realization of the library, at a level where there is a certain degree of statistical confidence (e.g., 95%) that any given member of the theoretical library is present in the physical realization of the library at least at a certain frequency (e.g., at least once, twice, three times, four times, five times, or more) in the library.

In a library, it is generally assumed that the number of copies of a given clone obeys a Poisson probability distribution (see Feller, W. An Introduction to Probability Theory and Its Applications, 1968, Wiley New York, incorporated by reference in its entirety). The probability of a Poisson random number being zero, corresponding to the probability of missing a given component member in an instance of a library (see below), is e N, where N is the average of the random number. For example, if there are 10⁶ possible theoretical members of a library and a physical realization of the library has 10⁷ members, with an equal probability of each member of the theoretical library being sampled, then the average number of times that each member occurs in the physical realization of the library is 10⁷/10⁶=10, and the probability that the number of copies of a given member is zero is e^(−N)=e⁻¹⁰=0.000045; or a 99.9955% chance that there is at least one copy of any of the 10⁶ theoretical members in this 10× oversampled library. For a 2.3× oversampled library one is 90% confident that a given component is present. For a 3× oversampled library one is 95% confident that a given component is present. For a 4.6× oversampled library one is 99% confident a given clone is present, and so on.

Therefore, if M is the maximum number of theoretical library members that can be feasibly physically realized, then M/3 is the maximum theoretical repertoire size for which one can be 95% confident that any given member of the theoretical library will be sampled. It is important to note that there is a difference between a 95% chance that a given member is represented and a 95% chance that every possible member is represented. In certain embodiments, the instant invention provides a rationally designed library with diversity so that any given member is 95% likely to be represented in a physical realization of the library. In other embodiments of the invention, the library is designed so that any given member is at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% likely to be represented in a physical realization of the library. For a review, see, e.g. Firth and Patrick, Biomol. Eng., 2005, 22: 105, and Patrick et al., Protein Engineering, 2003, 16: 451, each of which is incorporated by reference in its entirety.

In certain embodiments of the invention, a library may have a theoretical total diversity of X unique members and the physical realization of the theoretical total diversity may contain at least about 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, or more members. In some embodiments, the physical realization of the theoretical total diversity may contain about 1× to about 2×, about 2× to about 3×, about 3× to about 4×, about 4× to about 5×, about 5× to about 6× members. In other embodiments, the physical realization of the theoretical total diversity may contain about 1× to about 3×, or about 3× to about 5× total members.

An assumption underlying all directed evolution experiments is that the amount of molecular diversity theoretically possible is enormous compared with the ability to synthesize it, physically realize it, and screen it. The likelihood of finding a variant with improved properties in a given library is maximized when that library is maximally diverse. Patrick et al. used simple statistics to derive a series of equations and computer algorithms for estimating the number of unique sequence variants in libraries constructed by randomized oligonucleotide mutagenesis, error-prone PCR and in vitro recombination. They have written a suite of programs for calculating library statistics, such as GLUE, GLUE-IT, PEDEL, PEDEL-AA, and DRIVeR. These programs are described, with instructions on how to access them, in Patrick et al., Protein Engineering, 2003, 16: 451 and Firth et al., Nucleic Acids Res., 2008, 36: W281 (each of which is incorporated by reference in its entirety).

It is possible to construct a physical realization of a library in which some components of the theoretical diversity (such as CDRH3) are oversampled, while other aspects (VH/VL pairings) are not. For example, consider a library in which 10⁸ CDRH3 segments are designed to be present in a single VH chassis, and then paired with 10⁵ VL genes to produce 10¹³ (=10⁸*10⁵) possible full heterodimeric antibodies. If a physical realization of this library is constructed with a diversity of 10⁹ transformant clones, then the CDRH3 diversity is oversampled ten-fold (=10⁹/10³), however the possible VH/VL pairings are undersampled by 10⁻⁴ (=10⁹/10¹³). In this example, on average, each CDRH3 is paired only with 10 samples of the VL from the possible 10⁵ partners. In certain embodiments of the invention, it is the CDRH3 diversity that is preferably oversampled.

3.1. Other Variants of the Polynucleotide Sequences of the Invention

In certain embodiments, the invention relates to a polynucleotide that hybridizes with a polynucleotide taught herein, or that hybridizes with the complement of a polynucleotide taught herein. For example, an isolated polynucleotide that remains hybridized after hybridization and washing under low, medium, or high stringency conditions to a polynucleotide taught herein or the complement of a polynucleotide taught herein is encompassed by the present invention.

Exemplary low stringency conditions include hybridization with a buffer solution of about 30% to about 35% formamide, about 1 M NaCl, about 1% SDS (sodium dodecyl sulphate) at about 37° C., and a wash in about 1× to about 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at about 50° C. to about 55° C.

Exemplary moderate stringency conditions include hybridization in about 40% to about 45% formamide, about 1 M NaCl, about 1% SDS at about 37° C., and a wash in about 0.5× to about 1×SSC at abut 55° C. to about 60° C.

Exemplary high stringency conditions include hybridization in about 50% formamide, about 1 M NaCl, about 1% SDS at about 37° C., and a wash in about 0.1×SSC at about 60° C. to about 65° C.

Optionally, wash buffers may comprise about 0.1% to about 1% SDS.

The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

3.2. Sub-Libraries and Larger Libraries Comprising the Libraries or Sub-Libraries of the Invention

As described throughout the application, the libraries of the current invention are distinguished, in certain embodiments, by their human-like sequence composition and length, and the ability to generate a physical realization of the library which contains all members of (or, in some cases, even oversamples) a particular component of the library. Libraries comprising combinations of the libraries described herein (e.g., CDRH3 and CDRL3 libraries) are encompassed by the invention. Sub-libraries comprising portions of the libraries described herein are also encompassed by the invention (e.g. a CDRH3 library in a particular heavy chain chassis or a sub-set of the CDRH3 libraries). One of ordinary skill in the art will readily recognize that each of the libraries described herein has several components (e.g., CDRH3, VH, CDRL3, VL, etc.), and that the diversity of these components can be varied to produce sub-libraries that fall within the scope of the invention.

Moreover, libraries containing one of the libraries or sub-libraries of the invention also fall within the scope of the invention. For example, in certain embodiments of the invention, one or more libraries or sub-libraries of the invention may be contained within a larger library, which may include sequences derived by other means, for example, non-human or human sequence derived by stochastic or semi-stochastic synthesis. In certain embodiments of the invention, at least about 1% of the sequences in a polynucleotide library may be those of the invention (e.g. CDRH3 sequences, CDRL3 sequences, VH sequences, VL sequences), regardless of the composition of the other 99% of sequences. In other embodiments of the invention, at least about 0.001%, 0.01%, 0.1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91,%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequences in any polynucleotide library may be those of the invention, regardless of the composition of the other sequences. In some embodiments, the sequences of the invention may comprise about 0.001% to about 1%, about 1% to about 2%, about 2% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99% of the sequences in any polynucleotide library, regardless of the composition of the other sequences. Thus, libraries more diverse than one or more libraries or sub-libraries of the invention, but yet still comprising one or more libraries or sub-libraries of the invention, in an amount in which the one or more libraries or sub-libraries of the invention can be effectively screened and from which sequences encoded by the one or more libraries or sub-libraries of the invention can be isolated, also fall within the scope of the invention.

3.3. Alternative Scaffolds

In certain embodiments of the invention, the amino acid products of a library of the invention (e.g., a CDRH3 or CDRL3) may be displayed on an alternative scaffold. Several of these scaffolds have been shown to yield molecules with specificities and affinities that rival those of antibodies. Exemplary alternative scaffolds include those derived from fibronectin (e.g., AdNectin), the β-sandwich (e.g., iMab), lipocalin (e.g., Anticalin), EETI-IFAGRP, BPTIFLAC1-D1/IT1-D2 (e.g. Kunitz domain), thioredoxin (e.g., peptide aptamer), protein A (e.g., Affibody), ankyrin repeats (e.g., DARPin), γB-crystallin/ubiquitin (e.g., Affilin), CTLD₃ (e.g., Tetranectin), and (LDLR-A module)₃ (e.g., Avimers). Additional information on alternative scaffolds are provided in Binz et al., Nat. Biotechnol., 2005 23: 1257 and Skerra, Current Opin. in Biotech., 2007 18: 295-304, each of which is incorporated by reference in its entirety.

4. Other Embodiments of the Invention

In certain embodiments, the invention comprises a synthetic preimmune human antibody CDRH3 library comprising 10⁷ to 10⁸ polynucleotide sequences representative of the sequence diversity and length diversity found in known heavy chain CDR3 sequences.

In other embodiments, the invention comprises a synthetic preimmune human antibody CDRH3 library comprising polynucleotide sequences encoding CDRH3 represented by the following formula:

[G/D/E/-][N1][DH][N2][H3-JH],

wherein [G/D/E/-] is zero to one amino acids in length, [N1] is zero to three amino acids, [DH] is three to ten amino acids in length, [N2] is zero to three amino acids in length, and [H3-JH] is two to nine amino acids in length.

In certain embodiments of the invention, [G/D/E/-] is represented by an amino acid sequence selected from the group consisting of: G, D, E, and nothing.

In some embodiments of the invention, [N1] is represented by an amino acid sequence selected from the group consisting of: G, R, S, P, L, A, V, T, (G/P)(G/R/S/P/L/A/V/T), (R/S/L/A/V/T)(G/P), GG(G/R/S/P/L/A/V/T), G(R/S/P/L/A/V/T)G, (R/S/P/L/A/V/T)GG, and nothing.

In certain embodiments of the invention, [N2] is represented by an amino acid sequence selected from the group consisting of: G, R, S, P, L, A, V, T, (G/P)(G/R/S/P/L/A/V/T), (R/S/L/A/V/T)(G/P), GG(G/R/S/P/L/A/V/T), G(R/S/P/L/A/V/T)G, (R/S/P/L/A/V/T)GG, and nothing.

In some embodiments of the invention, [DH] comprises a sequence selected from the group consisting of: IGHD3-10 reading frame 1, IGHD3-10 reading frame 2, IGHD3-10 reading frame 3, IGHD3-22 reading frame 2, IGHD6-19 reading frame 1, IGHD6-19 reading frame 2, IGHD6-13 reading frame 1, IGHD6-13 reading frame 2, IGHD3-03 reading frame 3, IGHD2-02 reading frame 2, IGHD2-02 reading frame 3, IGHD4-17 reading frame 2, IGHD1-26 reading frame 1, IGHD1-26 reading frame 3, IGHD5-5/5-18 reading frame 3, IGHD2-15 reading frame 2, and all possible N-terminal and C-terminal truncations of the above-identified IGHDs down to three amino acids.

In certain embodiments of the invention, [H3-JH] comprises a sequence selected from the group consisting of: AEYFQH (SEQ ID NO: 17), EYFQH, YFQH, FQH, QH, YWYFDL (SEQ ID NO: 18), WYFDL, YFDL, FDL, DL, AFDV (SEQ ID NO: 19), FDV, DV, YFDY (SEQ ID NO: 20), FDY, DY, NWFDS (SEQ ID NO: 21), WFDS, FDS, DS, YYYYYGMDV (SEQ ID NO: 22), YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, MDV, and DV.

In some embodiments of the invention, the sequences represented by [G/D/E/-][N1][ext-DH][N2][H3-JH] comprise a sequence of about 3 to about 26 amino acids in length.

In certain embodiments of the invention, the sequences represented by [G/D/E/-][N1][ext-DH][N2][H3-JH] comprise a sequence of about 7 to about 23 amino acids in length.

In some embodiments of the invention, the library comprises about 10⁷ to about 10¹⁰ sequences.

In certain embodiments of the invention, the library comprises about 10⁷ sequences.

In some embodiments of the invention, the polynucleotide sequences of the libraries further comprise a 5′ polynucleotide sequence encoding a framework 3 (FRM3) region on the corresponding N-terminal end of the library sequence, wherein the FRM3 region comprises a sequence of about 1 to about 9 amino acid residues.

In certain embodiments of the invention, the FRM3 region comprises a sequence selected from the group consisting of CAR, CAK, and CAT.

In some embodiments of the invention, the polynucleotide sequences further comprise a 3′ polynucleotide sequence encoding a framework 4 (FRM4) region on the corresponding C-terminal end of the library sequence, wherein the FRM4 region comprises a sequence of about 1 to about 9 amino acid residues.

In certain embodiments of the invention, the library comprises a FRM4 region comprising a sequence selected from WGRG (SEQ ID NO: 23) and WGQG (SEQ ID NO: 23).

In some embodiments of the invention, the polynucleotide sequences further comprise an FRM3 region coding for a corresponding polypeptide sequence comprising a sequence selected from the group consisting of CAR, CAK, and CAT; and an FRM4 region coding for a corresponding polypeptide sequence comprising a sequence selected from WGRG (SEQ ID NO: 23) and WGQG (SEQ ID NO: 23).

In certain embodiments of the invention, the polynucleotide sequences further comprise 5′ and 3′ sequences which facilitate homologous recombination with a heavy chain chassis.

In some embodiments, the invention comprises a synthetic preimmune human antibody light chain library comprising polynucleotide sequences encoding human antibody kappa light chains represented by the formula:

[IGKV (1-95)][F/L/I/R/W/Y][JK].

In certain embodiments of the invention, [IGKV (1-95)] is selected from the group consisting of IGKV3-20 (1-95), IGKV1-39 (1-95), IGKV3-11 (1-95), IGKV3-15 (1-95), IGKV1-05 (1-95), IGKV4-01 (1-95), IGKV2-28 (1-95), IGKV 1-33 (1-95), IGKV1-09 (1-95), IGKV1-12 (1-95), IGKV2-30 (1-95), IGKV1-27 (1-95), IGKV1-16 (1-95), and truncations of said group up to and including position 95 according to Kabat.

In some embodiments of the invention, [F/L/J/R/W/Y] is an amino acid selected from the group consisting of F, L, I, R, W, and Y.

In certain embodiments of the invention, [JK] comprises a sequence selected from the group consisting of TFGQGTKVEIK (SEQ ID NO: 528) and TFGGGT (SEQ ID NO: 529).

In some embodiments of the invention, the light chain library comprises a kappa light chain library.

In certain embodiments of the invention, the polynucleotide sequences further comprise 5′ and 3′ sequences which facilitate homologous recombination with a light chain chassis.

In some embodiments, the invention comprises a method for producing a synthetic preimmune human antibody CDRH3 library comprising 10⁷ to 10⁸ polynucleotide sequences, said method comprising:

-   -   a) selecting the CDRH3 polynucleotide sequences encoded by the         CDRH3 sequences, as follows:         -   {0 to 5 amino acids selected from the group consisting of             fewer than ten of the amino acids preferentially encoded by             terminal deoxynucleotidyl transferase (TdT) and             preferentially functionally expressed by human B cells},             followed by         -   {all possible N or C-terminal truncations of IGHD alone and             all possible combinations of N and C-terminal truncations},             followed by         -   {0 to 5 amino acids selected from the group consisting of             fewer than ten of the amino acids preferentially encoded by             TdT and preferentially functionally expressed by human B             cells}, followed by         -   {all possible N-terminal truncations of IGHJ, down to DXWG,             wherein X is S, V, L, or Y}; and     -   b) synthesizing the CDRH3 library described in a) by chemical         synthesis, wherein a synthetic preimmune human antibody CDRH3         library is produced.

In certain embodiments, the invention comprises a synthetic preimmune human antibody CDRH3 library comprising 10⁷ to 10¹⁰ polynucleotide sequences representative of known human IGHD and IGHJ germline sequences encoding CDRH3, represented by the following formula:

-   -   {0 to 5 amino acids selected from the group consisting of fewer         than ten of the amino acids preferentially encoded by terminal         deoxynucleotidyl transferase (TdT) and preferentially         functionally expressed by human B cells}, followed by {all         possible N or C-terminal truncations of IGHD alone and all         possible combinations of N and C-terminal truncations}, followed         by     -   {0 to 5 amino acids selected from the group consisting of fewer         than ten of the amino acids preferentially encoded by TdT and         preferentially functionally expressed by human B cells},         followed by     -   {all possible N-terminal truncations of IGHJ, down to DXWG (SEQ         ID NO: 530), wherein X is S, V, L, or Y}.

In certain embodiments, the invention comprises a synthetic preimmune human antibody heavy chain variable domain library comprising 10⁷ to 10¹⁰ polynucleotide sequences encoding human antibody heavy chain variable domains, said library comprising:

-   -   a) an antibody heavy chain chassis, and     -   b) a CDRH3 repertoire designed based on the human IGHD and IGHJ         germline sequences, as follows:         -   {0 to 5 amino acids selected from the group consisting of             fewer than ten of the amino acids preferentially encoded by             terminal deoxynucleotidyl transferase (TdT) and             preferentially functionally expressed by human B cells},             followed by         -   {all possible N or C-terminal truncations of IGHD alone and             all possible combinations of N and C-terminal truncations},             followed by         -   {0 to 5 amino acids selected from the group consisting of             fewer than ten of the amino acids preferentially encoded by             TdT and preferentially functionally expressed by human B             cells}, followed by         -   {all possible N-terminal truncations of IGHJ, down to DXWG             (SEQ ID NO: 530), wherein X is S, V, L, or Y}.

In some embodiments of the invention, the synthetic preimmune human antibody heavy chain variable domain library is expressed as a full length chain selected from the group consisting of an IgG1 full length chain, an IgG2 full length chain, an IgG3 full length chain, and an IgG4 full length chain.

In certain embodiments of the invention, the human antibody heavy chain chassis is selected from the group consisting of IGHV4-34, IGHV3-23, IGHV5-51, IGHV1-69, IGHV3-30, IGHV4-39, IGHV1-2, IGHV1-18, IGHV2-5, IGHV2-70, IGHV3-7, IGHV6-1, IGHV1-46, IGHV3-33, IGHV4-31, IGHV4-4, IGHV4-61, and IGHV3-15.

In some embodiments of the invention, the synthetic preimmune human antibody heavy chain variable domain library comprises 10⁷ to 10¹⁰ polynucleotide sequences encoding human antibody heavy chain variable domains, said library comprising:

-   -   a) an antibody heavy chain chassis, and     -   b) a synthetic preimmune human antibody CDRH3 library.

In some embodiments of the invention, the polynucleotide sequences are single-stranded coding polynucleotide sequences.

In certain embodiments of the invention, the polynucleotide sequences are single-stranded non-coding polynucleotide sequences.

In some embodiments of the invention, the polynucleotide sequences are double-stranded polynucleotide sequences.

In certain embodiments, the invention comprises a population of replicable cells with a doubling time of four hours or less, in which a synthetic preimmune human antibody repertoire is expressed.

In some embodiments of the invention, the population of replicable cells are yeast cells.

In certain embodiments, the invention comprises a method of generating a full-length antibody library comprising transforming a cell with a preimmune human antibody heavy chain variable domain library and a synthetic preimmune human antibody light chain library.

In some embodiments, the invention comprises a method of generating a full-length antibody library comprising transforming a cell with a preimmune human antibody heavy chain variable domain library and a synthetic preimmune human antibody light chain library.

In certain embodiments, the invention comprises a method of generating an antibody library comprising synthesizing polynucleotide sequences by split-pool DNA synthesis.

In some embodiments of the invention, the polynucleotide sequences are selected from the group consisting of single-stranded coding polynucleotide sequences, single-stranded non-coding polynucleotide sequences, and double-stranded polynucleotide sequences.

In certain embodiments, the invention comprises a synthetic full-length preimmune human antibody library comprising about 10⁷ to about 10¹⁰ polynucleotide sequences representative of the sequence diversity and length diversity found in known heavy chain CDR3 sequences.

In certain embodiments, the invention comprises a method of selecting an antibody of interest from a human antibody library, comprising providing a synthetic preimmune human antibody CDRH3 library comprising a theoretical diversity of (N) polynucleotide sequences representative of the sequence diversity and length diversity found in known heavy chain CDR3 sequences, wherein the physical realization of that diversity is an actual library of a size at least 3(N), thereby providing a 95% probability that a single antibody of interest is present in the library, and selecting an antibody of interest.

In some embodiments of the invention, the theoretical diversity is about 10⁷ to about 10⁸ polynucleotide sequences.

EXAMPLES

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, PCR technology, immunology (especially, e.g., antibody technology), expression systems (e.g., yeast expression, cell-free expression, phage display, ribosome display, and PROFUSION™), and any necessary cell culture that are within the skill of the art and are explained in the literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); DNA Cloning, Vols. 1 and 2, (D. N. Glover, Ed. 1985); Oligonucleotide Synthesis (M. J. Gait, Ed. 1984); PCR Handbook Current Protocols in Nucleic Acid Chemistry, Beaucage, Ed. John Wiley & Sons (1999) (Editor); Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford Univ Press (1999); PCR Protocols: A Guide to Methods and Applications, Innis et al., Academic Press (1990); PCR Essential Techniques: Essential Techniques, Burke, Ed., John Wiley & Son Ltd (1996); The PCR Technique: RT-PCR, Siebert, Ed., Eaton Pub. Co. (1998); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992); Large-Scale Mammalian Cell Culture Technology, Lubiniecki, A., Ed., Marcel Dekker, Pub., (1990); Phage Display: A Laboratory Manual, C. Barbas (Ed.), CSHL Press, (2001); Antibody Phage Display, P O'Brien (Ed.), Humana Press (2001); Border et al., Nature Biotechnology, 1997, 15: 553; Border et al., Methods Enzymol., 2000, 328: 430; ribosome display as described by Pluckthun et al. in U.S. Pat. No. 6,348,315, and Profusion™ as described by Szostak et al. in U.S. Pat. Nos. 6,258,558; 6,261,804; and 6,214,553; and bacterial periplasmic expression as described in US20040058403A1. Each of the references cited in this paragraph is incorporated by reference in its entirety.

Further details regarding antibody sequence analysis using Kabat conventions and programs to screen aligned nucleotide and amino acid sequences may be found, e.g. in Johnson et al., Methods Mol. Biol., 2004, 248: 11; Johnson et al., Int. Immunol., 1998, 10: 1801; Johnson et al., Methods Mol. Biol., 1995, 51: 1; Wu et al., Proteins, 1993, 16: 1; and Martin, Proteins, 1996, 25: 130. Each of the references cited in this paragraph is incorporated by reference in its entirety.

Further details regarding antibody sequence analysis using Chothia conventions may be found, e.g., in Chothia et al., J. Mol. Biol., 1998, 278: 457; Morea et al., Biophys. Chem., 1997, 68: 9; Morea et al., J. Mol. Biol., 1998, 275: 269; Al-Lazikani et al., J. Mol. Biol., 1997, 273: 927. Barre et al, Nat. Struct. Biol., 1994, 1: 915; Chothia et al., J. Mol. Biol., 1992, 227: 799; Chothia et al., Nature, 1989, 342: 877; and Chothia et al., J. Mol. Biol., 1987, 196: 901. Further analysis of CDRH3 conformation may be found in Shirai et al., FEBS Lett., 1999, 455: 188 and Shirai et al., FEBS Lett., 1996, 399: 1. Further details regarding Chothia analysis are described, for example, in Chothia et al., Cold Spring Harb. Symp. Quant Biol., 1987, 52: 399. Each of the references cited in this paragraph is incorporated by reference in its entirety.

Further details regarding CDR contact considerations are described, for example, in MacCallum et al., J. Mol. Biol., 1996, 262: 732, incorporated by reference in its entirety.

Further details regarding the antibody sequences and databases referred to herein are found, e.g., in Tomlinson et al., J. Mol. Biol., 1992, 227: 776, VBASE2 (Retter et al., Nucleic Acids Res., 2005, 33: D671); BLAST (www.ncbi.nlm.nih.gov/BLAST/); CDHIT (bioinformatics.ljcrf.edu/cd-hi/); EMBOSS (www.hgmp.mrc.ac.uk/Software/EMBOSS/); PHYLIP (evolution.genetics.washington.edu/phylip.html); and FASTA (fasta.bioch.virginia.edu). Each of the references cited in this paragraph is incorporated by reference in its entirety.

Example 1 Design of an Exemplary VH_Chassis Library

This example demonstrates the selection and design of exemplary, non-limiting VH chassis sequences of the invention. VH chassis sequences were selected by examining collections of human IGHV germline sequences (Scaviner et al., Exp. Clin. Immunogenet., 1999, 16: 234; Tomlinson et al., J. Mol. Biol., 1992, 227: 799; Matsuda et al., J. Exp. Med., 1998, 188: 2151, each incorporated by reference in its entirety). As discussed in the Detailed Description, as well as below, a variety of criteria can be used to select VH chassis sequences, from these data sources or others, for inclusion in the library.

Table 3 (adapted from information provided in Scaviner et al., Exp. Clin. Immunogenet., 1999, 16: 234; Matsuda et al., J. Exp. Med., 1998, 188: 2151; and Wang et al. Immunol. Cell. Biol., 2008, 86: 111, each incorporated by reference in its entirety) lists the CDRH1 and CDRH2 length, the canonical structure and the estimated relative occurrence in peripheral blood, for the proteins encoded by each of the human IGHV germline sequences.

TABLE 3 IGHV Characteristics and Occurrence in Antibodies from Peripheral Blood Estimated Relative IGHV Length of Length of Canonical Occurrence in Germline CDRH1 CDRH2 Structures¹ Peripheral Blood² IGHV1-2 5 17 1-3 37 IGHV1-3 5 17 1-3 15 IGHV1-8 5 17 1-3 13 IGHV1-18 5 17 1-2 25 IGHV1-24 5 17 1-U 5 IGHV1-45 5 17 1-3 0 IGHV1-46 5 17 1-3 25 IGHV1-58 5 17 1-3 2 IGHV1-69 5 17 1-2 58 IGHV2-5 7 16 3-1 10 IGHV2-26 7 16 3-1 9 IGHV2-70 7 16 3-1 13 IGHV3-7 5 17 1-3 26 IGHV3-9 5 17 1-3 15 IGHV3-11 5 17 1-3 13 IGHV3-13 5 16 1-1 3 IGHV3-15 5 19 1-4 14 IGHV3-20 5 17 1-3 3 IGHV3-21 5 17 1-3 19 IGHV3-23 5 17 1-3 80 IGHV3-30 5 17 1-3 67 IGHV3-33 5 17 1-3 28 IGHV3-43 5 17 1-3 2 IGHV3-48 5 17 1-3 21 IGHV3-49 5 19 1-U 8 IGHV3-53 5 16 1-1 7 IGHV3-64 5 17 1-3 2 IGHV3-66 5 17 1-3 3 IGHV3-72 5 19 1-4 2 IGHV3-73 5 19 1-4 3 IGHV3-74 5 17 1-3 14 IGHV4-4 5 16 1-1 33 IGHV4-28 6 16 2-1 1 IGHV4-31 7 16 3-1 25 IGHV4-34 5 16 1-1 125 IGHV4-39 7 16 3-1 63 IGHV4-59 5 16 1-1 51 IGHV4-61 7 16 3-1 23 IGHV4-B 6 16 2-1 7 IGHV5-51 5 17 1-2 52 IGHV6-1 7 18 3-5 26 IGHV7-4-1 5 17 1-2 8 ¹Adapted from Chothia et al., J. Mol. Biol., 1992, 227: 799 ²Adapted from Table S1 of Wang et al., Immunol. Cell. Biol., 2008, 86: 111

In the currently exemplified library, 17 germline sequences were chosen for representation in the VH chassis of the library (Table 4). As described in more detail below, these sequences were selected based on their relatively high representation in the peripheral blood of adults, with consideration given to the structural diversity of the chassis and the representation of particular germline sequences in antibodies used in the clinic. These 17 sequences account for about 76% of the total sample of heavy chain sequences used to derive the results of Table 4. As outlined in the Detailed Description, these criteria are non-limiting, and one of ordinary skill in the art will readily recognize that a variety of other criteria can be used to select the VH chassis sequences, and that the invention is not limited to a library comprising the 17 VH chassis genes presented in Table 4.

TABLE 4 VH Chassis Selected for Use in the Exemplary Library Relative Length of Length of VH Chassis Occurrence CDRH1 CDRH2 Comment VH1-2 37 5 17 Among highest usage for VH1 family VH1-18 25 5 17 Among highest usage for VH1 family VH1-46 25 5 17 Among highest usage for VH1 family VH1-69 58 5 17 Highest usage for VH1 family. The four chosen VH1 chassis represent about 80% of the VH1 repertoire. VH3-7 26 5 17 Among highest usage in VH3 family VH3-15 14 5 19 Not among highest usage, but it has unique structure (H2 of length 19). Highest occurrence among those with such structure. VH3-23 80 5 17 Highest usage in VH3 family. VH3-30 67 5 17 Among highest usage in VH3 family VH3-33 28 5 17 Among highest usage in VH3 family VH3-48 21 5 17 Among highest usage in VH3 family. The six chosen VH3 chassis account for about 70% of the VH3 repertoire. VH4-31 25 7 16 Among highest usage in VH4 family VH4-34 125 5 16 Highest usage in VH4 family VH4-39 63 7 16 Among highest usage in VH4 family VH4-59 51 5 16 Among highest usage in VH4 family VH4-61 23 7 16 Among highest usage in VH4 family VH4-B 7 6 16 Not among highest usage in VH4 family, but has unique structure (H1 of length 6). The 6 chosen VH4 chassis account for close to 90% of the VH4 family repertoire VH5-51 52 5 17 High usage

In this particular embodiment of the library, VH chassis derived from sequences in the IGHV2, IGHV6 and IGHV7 germline families were not included. As described in the Detailed Description, this exemplification is not meant to be limiting, as, in some embodiments, it may be desirable to include one or more of these families, particularly as clinical information on antibodies with similar sequences becomes available, to produce libraries with additional diversity that is potentially unexplored, or to study the properties and potential of these IGHV families in greater detail. The modular design of the library of the present invention readily permits the introduction of these, and other, VH chassis sequences. The amino acid sequences of the VH chassis utilized in this particular embodiment of the library, which are derived from the IGHV germline sequences, are presented in Table 5. The details of the derivation procedures are presented below.

TABLE 5 Amino Acid Sequences for VH Chassis Selected for Inclusion in the Exemplary Library SEQ ID Chassis NO: FRM1 CDRH1 FRM2 CDRH2 FRM3 VH1-2 24 QVQLVQSG GYYMH WVRQAPG WINPNSG RVTMTRDTSI AEVKKPGA QGLEWMG GTNYAQK STAYMELSRL SVKVSCKA FQG RSDDTAVYYC SGYTFT AR VH1-18 25 QVQLVQSG SYGIS WVRQAPG WISAYNG RVTMTTDTST AEVKKPGA QGLEWMG NTNYAQK STAYMELRSL SVKVSCKA LQG RSDDTAVYYC SGYTFT AR VH1-46 26 QVQLVQSG SYYMH WVRQAPG IINPSGG RVTMTRDTST AEVKKPGA QGLEWMG STSYAQK STVYMELSSL SVKVSCKA FQC RSEDTAVYYC SGYTFT AR VH1-69 27 QVQLVQSG SYAIS WVRQAPG GIIPIFG RVTITADKST AEVKKPGS QGLEWMG TANYAQK STAYMELSSL SVKVSCKA FQG RSEDTAVYYC SGGTFS AR VH3-7 28 EVQLVESG SYWMS WVRQAPG NIKQDGS RFTISRDNAK GGLVQPGG KGLEWVA EKYYVDS NSLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AR VH3-15¹ 29 EVQLVESG NAWMS WVRQAPG RIKSKTD RFTISRDDSK GGLVKPGG KGLEWVG GGTTDYA NTLYLQMNSL SLRLSCAA APVKG RA EDTAVYYC SGFTFS AR VH3-23 30 EVQLLESG SYAMS WVRQAPG AISGSGG RFTISRDNSK GGLVQPGG KGLEWVS STYYADS NTLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AK VH3-30 31 QVQLVESG SYGMH WVRQAPG VISYDGS RFTISRDNSK GGVVQPGR KGLEWVA NKYYADS NTLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AR VH3-33 32 QVQLVESG SYGMH WVRQAPG VIWYDGS RFTISRDNSK GGVVQPGR KGLEWVA NKYYADS NTLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AR VH3-48 33 EVQLVESG SYSMN WVRQAPG YISSSSS RFTISRDNAK GGLVQPGG KGLEWVS TIYYADS NSLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AR VH4-31 34 QVQLQESG SGGYY WIRQHPG YIYYSGS RVTISVDTSK PGLVKPSQ WS KGLEWIG TYYNPSL NQFSLKLSSV TLSLTCTV KS TAADTAVYYC SGGSIS AR VH4-34² 35 QVQLQQWG GYYWS WIRQPPG EI DHS GS RVTISVDTSK AGLLKPSE KGLEWIG TNYNPSL NQFSLKLSSV TLSLTCAV KS TAADTAVYYC YGGSFS AR VH4-39 36 QLQLQESG SSSYY WIRQPPG SIYYSGS RVTISVDTSK PGLVKPSE WG KGLEWIG TYYNPSL NQFSLKLSSV TLSLTCTV KS TAADTAVYYC SGGSIS AR VH4-59 37 QVQLQESG SYYWS WIRQPPG YIYYSGS RVTISVDTSK PGLVKPSE KGLEWIG TNYNPSL NQFSLKLSSV TLSLTCTV KS TAADTAVYYC SGGSIS AR VH4-61 38 QVQLQESG SGSYY WIRQPPG YIYYSGS RVTISVDTSK PGLVKPSE WS KGLEWIG TNYNPSL NQFSLKLSSV TLSLTCTV KS TAADTAVYYC SGGSVS AR VH4-B 39 QVQLQESG SGYYW WIRQPPG SIYHSGS RVTISVDTSK PGLVKPSE G KGLEWIG TYYNPSL NQFSLKLSSV TLSLTCAV KS TAADTAVYYC SGYSIS AR VH5-51 40 EVQLVQSG SYWIG WVRQMPG IIYPGDS QVTISADKSI AEVKKPGE KGLEWMG DTRYSPS STAYLQWSSL SLKISCKG FQG KASDTAVYYC SGYSFT AR ¹The original KT sequence in VH3-15 was mutated to RA (bold/underlined) and TT to AR (bold/underlined), in order to match other VH3 family members selected for inclusion in the library. The modification to RA was made so that no unique sequence stretches of up to about 20 amino acids are created. Without being bound by theory, this modification is expected to reduce the odds of introducing novel T-cell epitopes in the VH3-15-derived chassis sequence. The avoidance of T cell epitopes is an additional criterion that can be considered in the design of certain libraries of the invention. ²The original NHS motif in VH4-34 was mutated to DHS, in order to remove a possible N-linked glycosylation site in CDR-H2. In certain embodiments of the invention, for example, if the library is transformed into yeast, this may prevent unwanted N-linked glycosylation.

Table 5 provides the amino acid sequences of the seventeen chassis. In nucleotide space, most of the corresponding germline nucleotide sequences include two additional nucleotides on the 3′ end (i.e., two-thirds of a codon). In most cases, those two nucleotides are GA. In many cases, nucleotides are added to the 3′ end of the IGHV-derived gene segment in vivo, prior to recombination with the IGHD gene segment. Any additional nucleotide would make the resulting codon encode one of the following two amino acids: Asp (if the codon is GAC or GAT) or Glu (if the codon is GAA or GAG). One, or both, of the two 3′-terminal nucleotides may also be deleted in the final rearranged heavy chain sequence. If only the A is deleted, the resulting amino acid is very frequently a G. If both nucleotides are deleted, this position is “empty,” but followed by a general V-D addition or an amino acid encoded by the IGHD gene. Further details are presented in Example 5. This first position, after the CAR or CAK motif at the C-terminus of FRM3 (Table 5), is designated the “tail.” In the currently exemplified embodiment of the library, this residue may be G, D, E, or nothing. Thus, adding the tail to any chassis enumerated above (Table 5) can produce one of the following four schematic sequences, wherein the residue following the VH chassis is the tail:

-   -   (1) [VH_Chassis]-[G]     -   (2) [VH_Chassis]-[D]     -   (3) [VH_Chassis]-[E]     -   (4) [VH_Chassis]

These structures can also be represented in the format:

[VH_Chassis]-[G/D/E/-],

wherein the hyphen symbol (−) indicates an empty or null position.

Using the CDRH3 numbering system defined in the Definitions section, the above sequences could be denoted to have amino acid 95 as G, D, or E, for instances (1), (2), and (3), respectively, while the sequence of instance 4 would have no position 95, and CDRH3 proper would begin at position 96 or 97.

In some embodiments of the invention, VH3-66, with canonical structure 1-1 (five residues in CDRH1 and 16 for CDRH2) may be included in the library. The inclusion of VH3-66 may compensate for the removal of other chassis from the library, which may not express well in yeast under some conditions (e.g., VH4-34 and VH4-59).

Example 2 Design of VH_Chassis Variants with Variation Within CDRH1 and CDRH2

This example demonstrates the introduction of further diversity into the VH chassis by creating mutations in the CDRH1 and CDRH2 regions of each chassis shown in Example 1. The following approach was used to select the positions and nature of the amino acid variation for each chassis: First, the sequence identity between rearranged human heavy chain antibody sequences was analyzed (Lee et al., Immunogenetics, 2006, 57: 917; Jackson et al., J. Immunol. Methods, 2007, 324: 26) and they were classified by the origin of their respective IGHV germline sequence. As an illustrative example, about 200 sequences in the data set exhibited greatest identity to the IGHV1-69 germline, indicating that they were likely to have been derived from IGHV1-69. Next, the occurrence of amino acid residues at each position within the CDRH1 and CDRH2 segments, in each germline family selected in Example 1 was determined. For VH1-69, these occurrences are illustrated in Tables 6 and 7. Second, neutral and/or smaller amino acid residues were favored, where possible, as replacements. Without being bound by theory, the rationale for the choice of these amino acid residues is the desire to provide a more flexible and less sterically hindered context for the display of a diversity of CDR sequences.

TABLE 6 Occurrence of Amino Acid Residues at Each Position Within IGHV1-69-derived CDRH1 Sequences 31 32 33 34 35 S Y A I S A 1 0 129  0 0 C 0 1 0 0 2 D 0 5 1 0 0 E 0 0 0 0 0 F 0 9 1 8 0 G 0 0 24  0 3 H 2 11  0 0 4 I 2 0 0 159  1 K 3 0 0 0 0 L 0 10  2 5 0 M 1 0 0 0 0 N 21  2 2 0 27  P 0 0 1 0 0 Q 1 1 0 0 5 R 9 0 0 0 1 S 133  3 7 0 129  T 12  1 10  0 12  V 0 0 7 13  0 W 0 0 0 0 0 Y 0 142  1 0 1

TABLE 7 Occurrence of Amino Acid Residues at Each Position Within IGHV1-69-derived CDRH2 Sequences 50 51 52 52A 53 54 55 56 57 G I I P I F G T A A 0 0 7 0 2 0 4 3 132  C 0 0 0 0 0 0 0 0 0 D 1 0 0 0 0 0 11  0 1 E 2 0 0 0 0 0 4 0 0 F 0 1 0 1 7 119  0 0 0 G 135  0 1 0 0 0 155  0 3 H 0 0 0 0 1 0 0 0 0 I 0 166  159  0 132  2 0 34  0 K 1 0 0 0 0 0 0 4 1 L 0 1 2 0 16  37  0 1 0 M 0 6 2 0 9 1 0 3 1 N 0 0 1 0 2 0 5 0 0 P 0 2 0 181  1 3 0 0 15  Q 0 0 0 0 0 0 1 0 1 R 44  0 0 0 0 0 1 4 0 S 1 0 1 1 2 6 3 5 8 T 1 1 7 2 2 1 0 127  15  V 0 8 5 0 11  4 0 4 8 W 0 0 0 0 0 1 0 0 0 Y 0 0 0 0 0 11  1 0 0 58 59 60 61 62 63 64 65 N Y A Q K F Q G A 0 0 178  0 0 0 0 0 C 0 0 0 0 0 0 0 0 D 21  0 0 0 2 0 0 12  E 2 0 1 1 4 0 2 0 F 0 0 0 0 0 180  0 0 G 1 0 0 0 0 0 0 173  H 4 4 0 3 0 0 4 0 I 2 1 0 0 0 0 0 0 K 5 0 0 2 156  0 3 0 L 0 0 0 0 0 3 2 0 M 0 0 0 0 0 0 0 0 N 132  1 0 0 8 0 0 0 P 0 0 3 6 0 0 0 0 Q 0 0 0 173  2 0 164  0 R 3 0 0 0 13  0 9 0 S 7 0 2 0 0 1 0 0 T 8 3 1 0 0 0 0 0 V 0 0 0 0 0 0 0 0 W 0 0 0 0 0 0 0 0 Y 0 176  0 0 0 1 1 0

The original germline sequence is provided in the second row of the tables, in bold font, beneath the residue number (Kabat system). The entries in the table indicate the number of times a given amino acid residue (first column) is observed at the indicated CDRH1 (Table 6) or CDRH2 (Table 7) position. For example, at position 33 the amino acid type G (glycine) is observed 24 times in the set of IGHV1-69-based sequences that were examined. Thus, applying the criteria above, variants were constructed with N at position 31, L at position 32 (H can be charged, under some conditions), G and T at position 33, no variants at position 34 and N at position 35, resulting in the following VH1-69 chassis CDRH1 single-amino acid variant sequences:

N YAIS (SEQ ID NO: 41) S L AIS (SEQ ID NO: 42) SY G IS (SEQ ID NO: 43) SY T IS (SEQ ID NO: 44) SYAI N (SEQ ID NO: 45)

Similarly, the analysis that produced Table 7 provided a basis for choosing the following single-amino acid variant sequences for VH1-69 chassis CDRH2s:

S IIPIFGTANYAQKFQG (SEQ ID NO: 46) GI A PIFGTANYAQKFQG (SEQ ID NO: 47) GIIPI L GTANYAQKFQG (SEQ ID NO: 48) GIIPIFGTA S YAQKFQG (SEQ ID NO: 49)

A similar approach was used to design and construct variants of the other selected chassis; the resulting CDRH1 and CDRH2 variants for each of the exemplary chassis are provided in Table 8. One of ordinary skill in the art will readily recognize that the methods described herein can be applied to create variants of other VH chassis and VL chassis.

TABLE 8 VH Chassis Variants SEQ ID SEQ ID Chassis CDRH1 NO: CDRH2 NO: 1-18.0 SYGIS 50 WISAYNGNT 56 NYAQKLQG 1-18.1 N YGIS 51 WISAYNGNT 56 NYAQKLQG 1-18.2 S N GIS 52 WISAYNGNT 56 NYAQKLQG 1-18.3 SY A IS 53 WISAYNGNT 56 NYAQKLQG 1-18.4 SYGI T 54 WISAYNGNT 56 NYAQKLQG 1-18.5 SYGI H 55 WISAYNGNT 56 NYAQKLQG 1-18.6 SYGIS 50 S ISAYNGNT 57 NYAQKLQG 1-18.7 SYGIS 50 WIS T YNGNT 58 NYAQKLQG 1-18.8 SYGIS 50 WIS P YNGNT 59 NYAQKLQG 1-18.9 SYGIS 50 WIS A YNGNT 60 YYAQKLQG 1-2.0 GYYMH 61 WINPNSGGT 67 NYAQKFQG 1-2.1 D YYMH 62 WINPNSGGT 67 NYAQKFQG 1-2.2 R YYMH 63 WINPNSGGT 67 NYAQKFQG 1-2.3 G S YMH 64 WINPNSGGT 67 NYAQKFQG 1-2.4 GY S MH 65 WINPNSGGT 67 NYAQKFQG 1-2.5 GYYM Q 66 WINPNSGGT 67 NYAQKFQG 1-2.6 GYYMH 61 S INPNSGGT 68 NYAQKFQG 1-2.7 GYYMH 61 WINP S SGGT 69 NYAQKFQG 1-2.8 GYYMH 61 WINPNSGGT 70 K YAQKFQG 1-2.9 GYYMH 61 WINPNSGGT 71 S YAQKFQG 1-46.0 SYYMH 72 IINPSGGST 79 SYAQKFQG 1-46.1 N YYMH 73 IINPSGGST 79 SYAQKFQG 1-46.2 S S YMH 74 IINPSGGST 79 SYAQKFQG 1-46.3 SY S MH 75 IINPSGGST 79 SYAQKFQG 1-46.4 SYY I H 76 IINPSGGST 79 SYAQKFQG 1-46.5 SYYM V 77 IINPSGGST 79 SYAQKFQG 1-46.6 SYYM S 78 IINPSGGST 79 SYAQKFQG 1-46.7 SYYMH 72 V INPSGGST 80 SYAQKFQG 1-46.8 SYYMH 72 IINP G GGST 81 SYAQKFQG 1-46.9 SYYMH 72 IINPSGGST 82 T YAQKFQG 1-69.0 SYAIS 83 GIIPIFGTA 84 NYAQKFQG 1-69.1 N YAIS 41 GIIPIFGTA 84 NYAQKFQG 1-69.2 S L AIS 42 GIIPIFGTA 84 NYAQKFQG 1-69.3 SY G IS 43 GIIPIFGTA 84 NYAQKFQG 1-69.4 SY T IS 44 GIIPIFGTA 84 NYAQKFQG 1-69.5 SYAI N 45 GIIPIFGTA 84 NYAQKFQG 1-69.6 SYAIS 83 S IIPIFGTA 46 NYAQKFQG 1-69.7 SYAIS 83 GI A PIFGTA 47 NYAQKFQG 1-69.8 SYAIS 83 GIIPI L GTA 48 NYAQKFQG 1-69.9 SYAIS 83 GIIPIFGTA 49 S YAQKFQG 3-15.0 NAWMS 85 RIKSKTDGG 91 TTDYAAPVK G 3-15.1 K AWMS 86 RIKSKTDGG 91 TTDYAAPVK G 3-15.2 D AWMS 87 RIKSKTDGG 91 TTDYAAPVK G 3-15.3 NA L MS 88 RIKSKTDGG 91 TTDYAAPVK G 3-15.4 NA A MS 89 RIKSKTDGG 91 TTDYAAPVK G 3-15.5 NAWM N 90 RIKSKTDGG 91 TTDYAAPVK G 3-15.6 NAWMS 85 S IKSKTDGG 92 TTDYAAPVK G 3-15.7 NAWMS 85 RIKS T TDGG 93 TTDYAAPVK G 3-15.8 NAWMS 85 RIKSK A DGG 94 TTDYAAPVK G 3-15.9 NAWMS 85 RIKSKTDGG 95 TT G YAAPVK G 3-23.0 SYAMS 96 AISGSGGST 100 YYADSVKG 3-23.1 N YAMS 97 AISGSGGST 100 YYADSVKG 3-23.2 T YAMS 98 AISGSGGST 100 YYADSVKG 3-23.3 S S AMS 99 AISGSGGST 100 YYADSVKG 3-23.4 SYAMS 96 G ISGSGGST 101 YYADSVKG 3-23.5 SYAMS 96 S ISGSGGST 102 YYADSVKG 3-23.6 SYAMS 96 T ISGSGGST 103 YYADSVKG 3-23.7 SYAMS 96 V ISGSGGST 104 YYADSVKG 3-23.8 SYAMS 96 AIS A SGGST 105 YYADSVKG 3-23.9 SYAMS 96 AISGSGGST 106 S YADSVKG 3-30.0 SYGMH 107 VISYDGSNK 111 YYADSVKG 3-30.1 NYGMH 108 VISYDGSNK 111 YYADSVKG 3-30.2 SY A MH 109 VISYDGSNK 111 YYADSVKG 3-30.3 SYG F H 110 VISYDGSNK 111 YYADSVKG 3-30.4 SYGMH 107 F ISYDGSNK 112 YYADSVKG 3-30.5 SYGMH 107 L ISYDGSNK 113 YYADSVKG 3-30.6 SYGMH 107 VIS S DGSNK 114 YYADSVKG 3-30.7 SYGMH 107 VISYDG N NK 115 YYADSVKG 3-30.8 SYGMH 107 VISYDGS I K 116 YYADSVKG 3-30.9 SYGMH 107 VISYDGSN Q 117 YYADSVKG 3-33.0 SYGMH 118 VIWYDGSNK 124 YYADSVKG 3-33.1 T YGMH 119 VIWYDGSNK 124 YYADSVKG 3-33.2 N YGMH 120 VIWYDGSNK 124 YYADSVKG 3-33.3 S S GMH 121 VIWYDGSNK 124 YYADSVKG 3-33.4 SY A MH 122 VIWYDGSNK 124 YYADSVKG 3-33.5 SYGM N 123 VIWYDGSNK 124 YYADSVKG 3-33.6 SYGMH 118 L IWYDGSNK 125 YYADSVKG 3-33.7 SYGMH 118 F IWYDGSNK 126 YYADSVKG 3-33.8 SYGMH 118 VIWYDGSNK 127 S YADSVKG 3-33.9 SYGMH 118 VIWYDGSNK 128 G YADSVKG 3-48.0 SYSMN 129 YISSSSSTI 136 YYADSVKG 3-48.1¹ N YSMN 130 YISSSSSTI 136 YYADSVKG 3-48.2 I YSMN 131 YISSSSSTI 136 YYADSVKG 3-48.3 S N SMN 132 YISSSSSTI 136 YYADSVKG 3-48.4 SY E MN 133 YISSSSSTI 136 YYADSVKG 3-48.5 SY N MN 134 YISSSSSTI 136 YYADSVKG 3-48.6 SYSM T 135 YISSSSSTI 136 YYADSVKG 3-48.7 SYSMN 129 TISSSSSTI 137 YYADSVKG 3-48.8 SYSMN 129 YISGSSSTI 138 YYADSVKG 3-48.9 SYSMN 129 YISSSSSTI 139 L YADSVKG 3-7.0 SYWMS 140 NIKQDGSEK 152 YYVDSVKG 3-7.1 T YWMS 141 NIKQDGSEK 152 YYVDSVKG 3-7.2 N YWMS 142 NIKQDGSEK 152 YYVDSVKG 3.7.3 S S WMS 143 NIKQDGSEK 152 YYVDSVKG 3-7.4 SY G MS 144 NIKQDGSEK 152 YYVDSVKG 3-7.5 SYWM T 145 NIKQDGSEK 152 YYVDSVKG 3-7.6 SYWMS 140 S IKQDGSEK 153 YYVDSVKG 3-7.7 SYWMS 140 NI N QDGSEK 154 YYVDSVKG 3-7.8 SYWMS 140 NIK S DGSEK 155 YYVDSVKG 3-7.9 SYWMS 140 NIKQDGSEK 156 Q YVDSVKG 4-31.0 SGGYYWS 147 YIYYSGSTY 157 YNPSLKS 4-31.1 SG S YYWS 148 YIYYSGSTY 157 YNPSLKS 4-31.2 SG T YYWS 149 YIYYSGSTY 157 YNPSLKS 4-31.3 SGG T YWS 150 YIYYSGSTY 157 YNPSLKS 4-31.4 SGGY S WS 151 YIYYSGSTY 157 YNPSLKS 4-31.5 SGGYYWS 147 S IYYSGSTY 158 YNPSLKS 4-31.6 SGGYYWS 147 N IYYSGSTY 159 YNPSLKS 4-31.7 SGGYYWS 147 YIYYSG N TY 160 YNPSLKS 4-31.8 SGGYYWS 147 YIYYSGST S 161 YNPSLKS 4-31.9 SGGYYWS 147 YIYYSGST V 162 YNPSLKS 4-34.0 GYYWS 163 EIDHSGSTN 166 YNPSLKS 4-34.1 D YYWS 164 EIDHSGSTN 166 YNPSLKS 4-34.2 GYYW T 165 EIDHSGSTN 166 YNPSLKS 4-34.3 GYYWS 163 D IDHSGSTN 167 YNPSLKS 4-34.4 GYYWS 163 EI S HSGSTN 168 YNPSLKS 4-34.5 GYYWS 163 EID Q SGSTN 169 YNPSLKS 4-34.6 GYYWS 163 EIDH G GSTN 170 YNPSLKS 4-34.7 GYYWS 163 EIDHSG N TN 171 YNPSLKS 4-34.8 GYYWS 163 EIDHSGST S 172 YNPSLKS 4-34.9 GYYWS 163 EIDHSGST D 173 YNPSLKS 4-39.0 SSSYYWG 174 SIYYSGSTY 181 YNPSLKS 4-39.1 T SSYYWG 175 SIYYSGSTY 181 YNPSLKS 4-39.2 S N SYYWG 176 SIYYSGSTY 181 YNPSLKS 4-39.3 SS D YYWG 177 SIYYSGSTY 181 YNPSLKS 4-39.4 SS N YYWG 178 SIYYSGSTY 181 YNPSLKS 4-39.5 SS R YYWG 179 SIYYSGSTY 181 YNPSLKS 4-39.6 SSSY A WG 180 SIYYSGSTY 181 YNPSLKS 4-39.7 SSSYYWG 174 N IYYSGSTY 182 YNPSLKS 4-39.8 SSSYYWG 174 SI S YSGSTY 183 YNPSLKS 4-39.9 SSSYYWG 174 SIYYSGST S 184 YNPSLKS 4-59.0 SYYWS 185 YIYYSGST N 189 YNPSLKS 4-59.1 T YYWS 186 YIYYSGSTN 189 YNPSLKS 4-59.2 S S YWS 187 YIYYSGSTN 189 YNPSLKS 4-59.3 SY S WS 188 YIYYSGSTN 189 YNPSLKS 4-59.4 SYYWS 185 F IYYSGSTN 190 YNPSLKS 4-59.5 SYYWS 185 H IYYSGSTN 191 YNPSLKS 4-59.6 SYYWS 185 S IYYSGSTN 192 YNPSLKS 4-59.7 SYYWS 185 YIY S SGSTN 193 YNPSLKS 4-59.8 SYYWS 185 YIYYSGST D 194 YNPSLKS 4-59.9 SYYWS 185 YIYYSGST T 195 YNPSLKS 4-61.0 SGSYYWS 196 YIYYSGSTN 202 YNPSLKS 4-61.1 SG G YYWS 197 YIYYSGSTN 202 YNPSLKS 4-61.2 SG N YYWS 198 YIYYSGSTN 202 YNPSLKS 4-61.3 SGS S YWS 199 YIYYSGSTN 202 YNPSLKS 4-61.4 SGSY S WS 200 YIYYSGSTN 202 YNPSLKS 4-61.5 SGSYYW T 201 YIYYSGSTN 202 YNPSLKS 4-61.6 SGSYYWS 196 R IYYSGSTN 203 YNPSLKS 4-61.7 SGSYYWS 196 S IYYSGSTN 204 YNPSLKS 4-61.8 SGSYYWS 196 YIY T SGSTN 205 YNPSLKS 4-61.9 SGSYYWS 196 YIYYSGST S 206 YNPSLKS 4-B.0 SGYYWG 207 SIYHSGSTY 212 YNPSLKS 4-B.1 S A YYWG 208 SIYHSGSTY 212 YNPSLKS 4-B.2 SG S YWG 209 SIYHSGSTY 212 YNPSLKS 4-B.3 SGY N WG 210 SIYHSGSTY 212 YNPSLKS 4-B.4 SGYYW A 211 SIYHSGSTY 212 YNPSLKS 4-B.5 SGYYWG 207 T IYHSGSTY 213 YNPSLKS 4-B.6 SGYYWG 207 S S YHSGSTY 214 YNPSLKS 4-B.7 SGYYWG 207 SIYHSG N TY 215 YNPSLKS 4-B.8 SGYYWG 207 SIYHSGST N 216 YNPSLKS 4-B.9 SGYYWG 207 SIYHSGST G 217 YNPSLKS 5-51.0 SYWIG 218 IIYPGDSDT 224 RYSPSFQG 5-51.1 T YWIG 219 IIYPGDSDT 224 RYSPSFQG 5-51.2 N YWIG 220 IIYPGDSDT 224 RYSPSFQG 5-51.3 S N WIG 221 IIYPGDSDT 224 RYSPSFQG 5-51.4 SY Y IG 222 IIYPGDSDT 224 RYSPSFQG 5-51.5 SYWI S 223 IIYPGDSDT 224 RYSPSFQG 5-51.6 SYWIG 218 S IYPGDSDT 225 RYSPSFQG 5-51.7 SYWIG 218 IIYP A DSDT 226 RYSPSFQG 5-51.8 SYWIG 218 IIYPGDS S T 227 RYSPSFQG 5-51.9 SYWIG 218 IIYPGDSDT 228 T YSPSFQG ¹Contains an N-linked glycosylation site which can be removed, if desired, as described herein.

As specified in the Detailed Description, other criteria can be used to select which amino acids are to be altered and the identity of the resulting altered sequence. This is true for any heavy chain chassis sequence, or any other sequence of the invention. The approach outlined above is meant for illustrative purposes and is non-limiting.

Example 3 Design of an Exemplary VK_Chassis Library

This example describes the design of an exemplary VK chassis library. One of ordinary skill in the art will recognize that similar principles may be used to design a Vλ library, or a library containing both VK and Vλ chassis. Design of a Vλ chassis library is presented in Example 4.

As was previously demonstrated in Example 1, for IGHV germline sequences, the sequence characteristics and occurrence of human IGKV germline sequences in antibodies from peripheral blood were analyzed. The data are presented in Table 9.

TABLE 9 IGKV Gene Characteristics and Occurrence in Antibodies from Peripheral Blood Estimated Relative Alternative CDRL1 CDRL2 Canonical Occurrence in IGKV Gene Names Length Length Structures¹ Peripheral Blood² IGKV1-05 L12 11 7 2-1-(U) 69  IGKV1-06 L11 11 7 2-1-(1) 14  IGKV1-08 L9 11 7 2-1-(1) 9 IGKV1-09 L8 11 7 2-1-(1) 24  IGKV1-12 L5, L19 11 7 2-1-(1) 32  IGKV1-13 L4, L18 11 7 2-1-(1) 13  IGKV1-16 L1 11 7 2-1-(1) 15  IGKV1-17 A30 11 7 2-1-(1) 34  IGKV1-27 A20 11 7 2-1-(1) 27  IGKV1-33 O8, O18 11 7 2-1-(1) 43  IGKV1-37 O14, O4 11 7 2-1-(1) 3 IGKV1-39 O2, O12 11 7 2-1-(1) 147  IGKV1D-16 L15 11 7 2-1-(1) 6 IGKV1D-17 L14 11 7 2-1-(1) 1 IGKV1D-43 L23 11 7 2-1-(1) 1 IGKV1D-8 L24 11 7 2-1-(1) 1 IGKV2-24 A23 16 7 4-1-(1) 8 IGKV2-28 A19, A3 16 7 4-1-(1) 62  IGKV2-29 A18 16 7 4-1-(1) 6 IGKV2-30 A17 16 7 4-1-(1) 30  IGKV2-40 O1, O11 17 7 3-1-(1) 3 IGKV2D-26 A8 16 7 4-1-(1) 0 IGKV2D-29 A2 16 7 4-1-(1) 20  IGKV2D-30 A1 16 7 4-1-(1) 4 IGKV3-11 L6 11 7 2-1-(1) 87  IGKV3-15 L2 11 7 2-1-(1) 53  IGKV3-20 A27 12 7 6-1-(1) 195  IGKV3D-07 L25 12 7 6-1-(1) 0 IGKV3D-11 L20 11 7 2-1-(U) 0 IGKV3D-20 A11 12 7 6-1-(1) 2 IGKV4-1 B3 17 7 3-1-(1) 83  IGKV5-2 B2 11 7 2-1-(1) 1 IGKV6-21 A10, A26 11 7 2-1-(1) 6 IGKV6D-41 A14 11 7 2-1-(1) 0 ¹Adapted from Tomlinson et al. EMBO J., 1995, 14: 4628, incorporated by reference in its entirety. The number in parenthesis refers to canonical structures in CDRL3, if one assuming the most common length (see Example 5 for further detail about CDRL3). ²Estimated from sets of human VK sequences compiled from the NCBI database; full set of GI numbers provided in Appendix A.

The 14 most commonly occurring IGKV germline genes (bolded in column 6 of Table 9) account for just over 90% of the usage of the entire repertoire in peripheral blood. From the analysis of Table 9, ten IGKV germline genes were selected for representation as chassis in the currently exemplified library (Table 10). All but V1-12 and V1-27 are among the top 10 most commonly occurring. IGKV germline genes VH2-30, which was tenth in terms of occurrence in peripheral blood, was not included in the currently exemplified embodiment of the library, in order to maintain the proportion of chassis with short (i.e., 11 or 12 residues in length) CDRL1 sequences at about 80% in the final set of 10 chassis. V1-12 was included in its place. V1-17 was more similar to other members of the V1 family that were already selected; therefore, V1-27 was included, instead of V1-17. In other embodiments, the library could include 12 chassis (e.g., the ten of Table 10 plus V1-17 and V2-30), or a different set of any “N” chassis, chosen strictly by occurrence (Table 9) or any other criteria. The ten chosen VK chassis account for about 80% of the usage in the data set believed to be representative of the entire kappa light chain repertoire.

TABLE 10 VK Chassis Selected for Use in the Exemplary Library Estimated Relative CDR-L1 CDR-L2 Canonical Occurrence in Chassis Length Length Structures Peripheral Blood VK1-5 11 7 2-1-(U) 69 VK1-12 11 7 2-1-(1) 32 VK1-27 11 7 2-1-(1) 27 VK1-33 11 7 2-1-(1) 43 VK1-39 11 7 2-1-(1) 147 VK2-28 16 7 4-1-(1) 62 VK3-11 11 7 2-1-(1) 87 VK3-15 11 7 2-1-(1) 53 VK3-20 12 7 6-1-(1) 195 VK4-1 17 7 3-1-(1) 83

The amino acid sequences of the selected VK chassis enumerated in Table 10 are provided in Table 11.

TABLE 11 Amino Acid Sequences for VK Chassis Selected for Inclusion in the Exemplary Library SEQ ID Chassis FRM1 CDRL1 FRM2 CDRL2 FRM3 CDRL3¹ NO: VK1-5 DIQMTQS RASQSI WYQQKP DASSLE GVPSRFSGSGSGT QYNSY 229 PSTLSAS SSWLA GKAPKL S EFTLTISSLQPDD S VGDRVTI LIY FATYYC TC VK1-12 DIQMTQS RASQGI WYQQKP AASSLQ GVPSRFSGSGSGT QANSF 230 PSSVSAS SSWLA GKAPKL S DFTLTISSLQPED P VGDRVTI LIY FATYYC TC VK1-27 DIQMTQS RASQGI WYQQKP AASTLQ GVPSRFSGSGSGT KYNSA 231 PSSLSAS SNYLA GKVPKL S DFTLTISSLQPED P VGDRVTI LIY VATYYC TC VK1-33 DIQMTQS QASQDI WYQQKP DASNLE GVPSRFSGSGSGT QYDNL 232 PSSLSAS SNYLN GKAPKL T DFTFTISSLQPED P VGDRVTI LIY IATYYC TC VK1-39 DIQMTQS RASQSI WYQQKP AASSLQ GVPSRFSGSGSGT QSYST 233 PSSLSAS SSYLN GKAPKL S DFTLTISSLQPED P VGDRVTI LIY FATYYC TC VK2-28 DIVMTQS RSSQSL WYLQKP LGSNRA GVPDRFSGSGSGT QALQT 234 PLSLPVT LHSNGY GQSPQL S DFTLKISRVEAED P PGEPASI NYLD LIY VGVYYC SC VK3-11 EIVLTQS RASQSV WYQQKP DASNRA GIPARFSGSGSGT QRSNW 235 PATLSLS SSYLA GQAPRL T DFTLTISSLEPED P PGERATL LIY FAVYYC SC VK3-15 EIVMTQS RASQSV WYQQKP GASTRA GIPARFSGSGSGT QYNNW 236 PATLSVS SSNLA GQAPRL T EFTLTISSLQSED P PGERATL LIY FAVYYC SC VK3-20 EIVLTQS RASQSV WYQQKP GASSRA GIPDRFSGSGSGT QYGSS 237 PGTLSLS SSSYLA GQAPRL T DFTLTISRLEPED P PGERATL LIY FAVYYC SC VK4-1 DIVMTQS KSSQSV WYQQKP WASTRE GVPDRFSGSGSGT QYYST 238 PDSLAVS LYSSNN GQPPKL S DFTLTISSLQAED P LGERATI KNYLA LIY VAVYYC NC ¹Note that the portion of the IGKV gene contributing to VKCDR3 is not considered part of the chassis as described herein. The VK chassis is defined as Kabat residues 1 to 88 of the IGKV-encoded sequence, or from the start of FRM1 to the end of FRM3. The portion of the VKCDR3 sequence contributed by the IGKV gene is referred to herein as the L3-VK region

Example 4 Design of an Exemplary Vλ Chassis Library

This example, describes the design of an exemplary Vλ chassis library. As was previously demonstrated in Examples 1-3, for the VH and VK chassis sequences, the sequence characteristics and occurrence of human IgλV germline-derived sequences in peripheral blood were analyzed. As with the assignment of other sequences set forth herein to germline families, assignment of VP sequences to a germline family was performed via SoDA and VBASE2 (Volpe and Kepler, Bioinformatics, 2006, 22: 438; Mollova et al., BMS Systems Biology, 2007, 1S: P30, each incorporated by reference in its entirety). The data are presented in Table 12.

TABLE 12 IGλV Gene Characteristics and Occurrence in Peripheral Blood Contribution of Estimated Relative IGλV Alternative Canonical IGVλ Gene to Occurrence in Gene Name Structures¹ CDRL3 Peripheral Blood² IGλV3-1 3R 11-7(*) 8 11.5 IGλV3-21 3H 11-7(*) 9 10.5 IGλV2-14 2A2 14-7(A) 9 10.1 IGλV1-40 1E 14-7(A) 9 7.7 IGλV3-19 3L 11-7(*) 9 7.6 IGλV1-51 1B 13-7(A) 9 7.4 IGλV1-44 1C 13-7(A) 9 7.0 IGλV6-57 6A 13-7(B) 7 6.1 IGλV2-8 2C 14-7(A) 9 4.7 IGλV3-25 3M 11-7(*) 9 4.6 IGλV2-23 2B2 14-7(A) 9 4.3 IGλV3-10 3P 11-7(*) 9 3.4 IGλV4-69 4B 12-11(*) 7 3.0 IGλV1-47 1G 13-7(A) 9 2.9 IGλV2-11 2E 14-7(A) 9 1.3 IGλV7-43 7A 14-7(B) 8 1.3 IGλV7-46 7B 14-7(B) 8 1.1 IGλV5-45 5C 14-11(*) 8 1.0 IGλV4-60 4A 12-11(*) 7 0.7 IGλV10-54 8A 14-7(B) 8 0.7 IGλV8-61 10A 13-7(C) 9 0.7 IGλV3-9 3J 11-7(*) 8 0.6 IGλV1-36 1A 13-7(A) 9 0.4 IGλV2-18 2D 14-7(A) 9 0.3 IGλV3-16 3A 11-7(*) 9 0.2 IGλV3-27 11-7(*) 7 0.2 IGλV4-3 5A 14-11(*) 8 0.2 IGλV5-39 4C 12-11(*) 12 0.2 IGλV9-49 9A 12-12(*) 12 0.2 IGλV3-12 3I 11-7(*) 9 0.1 ¹Adapted from Williams et al. J. Mol. Biol. 1996: 264, 220-32. The (*) indicates that the canonical structure is entirely defined by the lengths of CDRs L1 and L2. When distinct structures are possible for identical L1 and L2 length combinations, the structure present in a given gene is set forth as A, B, or C. ²Estimated from a set of human Vλ sequences compiled from the NCBI database; full set of GI codes set forth in Appendix B.

To choose a subset of the sequences from Table 12 to serve as chassis, those represented at less than 1% in peripheral blood (as extrapolated from analysis of published sequences corresponding to the GI codes provided in Appendix B) were first discarded. From the remaining 18 germline sequences, the top occurring genes for each unique canonical structure and contribution to CDRL3, as well as any germline gene represented at more than the 5% level, were chosen to constitute the exemplary VA chassis. The list of 11 such sequences is given in Table 13, below. These 11 sequences represent approximately 73% of the repertoire in the examined data set (Appendix B).

TABLE 13 Vλ Chassis Selected for Use in the Exemplary Library Canonical Relative Chassis CDRL1 Length CDRL2 Length Structure Occurrence Vλ3-1 11 7 11-7(*) 11.5 Vλ3-21 11 7 11-7(*) 10.5 Vλ2-14 14 7 14-7(A) 10.1 Vλ1-40 14 7 14-7(A) 7.7 Vλ3-19 11 7 11-7(*) 7.6 Vλ1-51 13 7 13-7(A) 7.4 Vλ1-44 13 7 13-7(A) 7.0 Vλ6-57 13 7 13-7(B) 6.1 Vλ4-69 12 11 12-11(*) 3.0 Vλ7-43 14 7 14-7(B) 1.3 Vλ5-45 11 11 14-11(*) 1.0

The amino acid sequences of the selected VP chassis enumerated in Table 13 are provided in Table 14, below.

TABLE 14 Amino Acid Sequences for Vλ Chassis Selected for Inclusion in the Exemplary Library Chassis FRM1 CDRL1 FRM2 CDRL2 FRM3 CDRL3² Vλ1-40 QSVLTQP TGSSSN WYQQLP GN---- GVPDRFSGSKSG-- QSYDSS SEQ ID PSVSGAP IGAGYD GTAPKL SNRPS TSASLAITGLQAEDE LSG NO: 531 GQRVTIS ---VH LIY ADYYC C Vλ1-44 QSVLTQP SGSSSN WYQQLP SN---- GVPDRFSGSKSG-- AAWDDS SEQ ID PSASGTP IGSNT- GTAPKL NQRPS TSASLAISGLQSEDE LNG NO: 532 GQRVTIS ---VN LIY ADYYC C Vλ1-51 QSVLTQP SGSSSN WYQQLP DN---- GIPDRFSGSKSG-- GTWDSS SEQ ID PSVSAAP IGNNY- GTAPKL NKRPS TSATLGITGLQTGDE LSA NO: 533 GQKVTIS ---VS LIY ADYYC C Vλ2-14 QSALTQP TGTSSD WYQQHP EV---- GVSNRFSGSKSG-- SSYTSS SEQ ID ASVSGSP VGGYNY GKAPKL SNRPS NTASLTISGLQAEDE STL NO: 534 GQSITIS ---VS MIY ADYYC C Vλ3-1¹ SYELTQP SGDKLG WYQQKP QD---- GIPERFSGSNSG-- QAWDSS SEQ ID PSVSVSP DKY--- GQSPVL SKRPS NTATLTISGTQAMDE TA- NO: 535 GQTASIT ---A S VIY ADYYC C Vλ3-19 SSELTQD QGDSLR WYQQKP GK---- GIPDRFSGSSSG-- NSRDSS SEQ ID PAVSVAL SYY--- GQAPVL NNRPS NTASLTITGAQAEDE GNH NO: 536 GQTVRIT ---AS VIY ADYYC C Vλ3-21 SYVLTQP GGNNIG WYQQKP YD---- GIPERFSGSNSG-- QVWDSS SEQ ID PSVSVAP SKS--- GQAPVL SDRPS NTATLTISRVEAGDE SDH NO: 537 GKTARIT ---VH VIY ADYYC C Vλ4-69 QLVLTQS TLSSGH WHQQQP LNSDGS GIPDRFSGSSSG-- QTWGTG SEQ ID PSASASL SSYA-- EKGPRY HSKGD AERYLTISSLQSEDE I-- NO: 538 GASVKLT ---IA LMK ADYYC C Vλ6-57 NFMLTQP TRSSGS WYQQRP ED---- GVPDRFSGSIDSSSN QSYDSS SEQ ID HSVSESP IASNY- GSSPTT NQRPS SASLTISGLKTEDEA N-- NO: 539 GKTVTIS ---VQ VIY DYYC C Vλ5-45 QAVLTQP TLRSGI WYQQKP YKSDSD GVPSRFSGSKDASAN MIWHSS SEQ ID ASLSASP NVGTYR GSPPQY KQQGS AGILLISGLQSEDEA AS- NO: 540 GASASLT ---IY LLR DYYC C Vλ7-43 QTVVTQE ASSTGA WFQQKP ST---- WTPARFSGSLLG-- LLYYGG SEQ ID PSLTVSP VTSGYY GQAPRA SNKHS GKAALTLSGVQPEDE AQ- NO: 541 GGTVTLT ---PN LIY AEYYC C ¹The last amino acid in CDRL1 of the Vλ3-1 chassis, S, differs from the corresponding one in the IGλV3-1 germline gene, C. This was done to avoid having a potentially unpaired CYS (C) amino acid in the resulting synthetic light chain. ²Note that, as for the VK chassis, the portion of the IGλV gene contributing to VλCDR3 is not considered part of the chassis as described herein. The Vλ chassis is defined as Kabat residues 1 to 88 of the IGλV-encoded sequence, or from the start of FRM1 to the end of FRM3. The portion of the VλCDR3 sequence contributed by the IGλV gene is referred to herein as the L3-Vλ region.

Example 5 Design of a CDRH3 Library

This example describes the design of a CDHR3 library from its individual components. In nature, the CDRH3 sequence is derived from a complex process involving recombination of three different genes, termed IGHV, IGHD and IGHJ. In addition to recombination, these genes may also undergo progressive nucleotide deletions: from the 3′ end of the IGHV gene, either end of the IGHD gene, and/or the 5′ end of the IGHJ gene. Non-templated nucleotide additions may also occur at the junctions between the V, D and J sequences. Non-templated additions at the V-D junction are referred to as “N1”, and those at the D-J junction are referred to as “N2”. The D gene segments may be read in three forward and, in some cases, three reverse reading frames.

In the design of the present exemplary library, the codon (nucleotide triplet) or single amino acid was designated as a fundamental unit, to maintain all sequences in the desired reading frame. Thus, all deletions or additions to the gene segments are carried out via the addition or deletion of amino acids or codons, and not single nucleotides. According to the CDRH3 numbering system of this application, CDRH3 extends from amino acid number 95 (when present; see Example 1) to amino acid 102.

Example 5.1 Selection of the DH Segments

In this illustrative example, selection of DH gene segments for use in the library was performed according to principles similar to those used for the selection of the chassis sequences. First, an analysis of IGHD gene usage was performed, using data from Lee et al, Immunogenetics, 2006, 57: 917; Corbett et al., PNAS, 1982, 79: 4118; and Souto-Carneiro et al., J. Immunol., 2004, 172: 6790 (each incorporated by reference in its entirety), with preference for representation in the library given to those IGHD genes most frequently observed in human sequences. Second, the degree of deletion on either end of the IGHD gene segments was estimated by comparison with known heavy chain sequences, using the SoDA algorithm (Volpe et al., Bioinformatics, 2006, 22: 438, incorporated by reference in its entirety) and sequence alignments. For the presently exemplified library, progressively deleted DH segments, as short as three amino acids, were included. As enumerated in the Detailed Description, other embodiments of the invention comprise DH segments with deletions to a different length, for example, about 1, 2, 4, 5, 6, 7, 8, 9, or 10 amino acids. Table 15 shows the relative occurrence of IGHD gene usage in human antibody heavy chain sequences isolated mainly from peripheral blood B cells (list adapted from Lee et al., Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety).

TABLE 15 Usage of IGHD Genes Based on Relative Occurrence in Peripheral Blood* Estimated Relative Occurrence IGHD Gene in Peripheral Blood³ IGHD3-10 117 IGHD3-22 111 IGHD6-19 95 IGHD6-13 93 IGHD3-3 82 IGHD2-2 63 IGHD4-17 61 IGHD1-26 51 IGHD5-5/5-18¹ 49 IGHD2-15 47 IGHD6-6 38 IGHD3-9 32 IGHD5-12 29 IGHD5-24 29 IGHD2-21 28 IGHD3-16 18 IGHD4-23 13 IGHD1-1 9 IGHD1-7 9 IGHD4-4/4-11² 7 IGHD1-20 6 IGHD7-27 6 IGHD2-8 4 IGHD6-25 3 ¹Although distinct genes in the genome, the nucleotide sequences of IGHD5-5 and IGHD5-18 are 100% identical and thus indistinguishable in rearranged VH sequences. ²IGHD4-4 and IGHD4-11 are also 100% identical. ³Adapted from Lee et al. Immunogenetics, 2006, 57: 917, by merging the information for distinct alleles of the same IGHD gene. *IGHD1-14 may also be included in the libraries of the invention.

The translations of the ten most commonly expressed IGHD gene sequences found in naturally occurring human antibodies, in three reading frames, are shown in Table 16. Those reading frames which occur most commonly in peripheral blood have been highlighted in gray. As in Table 15, data regarding IGHD sequence usage and reading frame statistics were derived from Lee et al., 2006, and data regarding IGHD sequence reading frame usage were further complemented by data derived from Corbett et al., PNAS, 1982, 79: 4118 and Souto-Carneiro et al., J. Immunol, 2004, 172: 6790, each of which is incorporated by reference in its entirety.

TABLE 16 Translations of the Ten Most Common Naturally Occurring IGHD Sequences, in Three Reading Frames (RF)

# represents a stop codon. Reading frames highlighted in gray correspond to the most commonly used reading frames.

In the presently exemplified library, the top 10 IGHD genes most frequently used in heavy chain sequences occurring in peripheral blood were chosen for representation in the library. Other embodiments of the library could readily utilize more or fewer D genes. The amino acid sequences of the selected IGHD genes, including the most commonly used reading frames and the total number of variants after progressive N- and C-terminal deletion to a minimum of three residues, are listed in Table 17. As depicted in Table 17, only the most commonly occurring alleles of certain IGHD genes were included in the illustrative library. This is, however, not required, and other embodiments of the invention may utilize IGHD reading frames that occur less frequently in the peripheral blood.

TABLE 17 D Genes Selected for use in the Exemplary Library IGHD Amino Acid SEQ ID Total Number of Gene¹ Sequence NO: Variants² IGHD1-26_1 GIVGATT 13 15 IGHD1-26_3 YSGSYY 14 10 IGHD2-2_2 GYCSSTSCYT 10   9³ IGHD2-2_3 DIVVVPAAM 11 28 IGHD2-15_2 GYCSGGSCYS 16  9 IGHD3-3_3 ITIFGVVII 9 28 IGHD3-10_1 VLLWFGELL 1 28 IGHD3-10_2 YYYGSGSYYN 2 36 IGHD3-10_3 ITMVRGVII 3 28 IGHD3-22_2 YYYDSSGYYY 4 36 IGHD4-17_2 DYGDY 12  6 IGHD5-5_3 GYSYGY 15 10 IGHD6-13_1 GYSSSWY 7 15 IGHD6-13_2 GIAAAG 8 10 IGHD6-19_1 GYSSGWY 5 15 IGHD6-19_2 GIAVAG 6 10 ¹The reading frame (RF) is specified _RF after the name of the gene. ²In most cases the total number of variants is given by (N − 1) times (N − 2) divided by two, where N is the total length in amino acids of the intact D segment. ³As detailed herein, the number of variants for segments containing a putative disulfide bond (two C or Cys residues) is limited in this illustrative embodiment.

For each of the selected sequences of Table 17, variants were generated by systematic deletion from the N- and/or C-termini, until there were three amino acids remaining. For example, for the IGHD4-17_(—)2 above, the full sequence DYGDY (SEQ ID NO: 12) may be used to generate the progressive deletion variants: DYGD, YGDY, DYG, GDY and YGD. In general, for any full-length sequence of size N, there will be a total of (N−1)*(N−2)/2 total variants, including the original full sequence. For the disulfide-loop-encoding segments, as exemplified by reading frame 2 of both IGHD2-2 and IGHD2-15, (i.e., IGHD2-2_(—)2 and IGH2-15_(—)2), the progressive deletions were limited, so as to leave the loop intact i.e., only amino acids N-terminal to the first Cys, or C-terminal to the second Cys, were deleted in the respective DH segment variants. The foregoing strategy was used to avoid the presence of unpaired cysteine residues in the exemplified version of the library. However, as discussed in the Detailed Description, other embodiments of the library may include unpaired cysteine residues, or the substitution of these cysteine residues with other amino acids. In the cases where the truncation of the IGHD gene is limited by the presence of the Cys residues, only 9 variants (including the original full sequence) were generated; e.g. for IGHD2-2_(—)2, the variants would be: GYCSSTSCYT (SEQ ID NO: 10), GYCSSTSCY, YCSSTSCYT, CSSTSCYT, GYCSSTSC, YCSSTSCY, CSSTSCY, YCSSTSC and CSSTSC.

According to the criteria outlined above, 293 DH sequences were obtained from the selected IGHD gene segments, including the original IGHD gene segments. Certain sequences are redundant. For example, it is possible to obtain the YYY variant from either IGHD3-10_(—)2 (full sequence YYYGSGSYYN (SEQ ID NO: 2)), or in two different ways from IGHD3-22_(—)2 (SEQ ID NO: 4) (YYYDSSGYYY). When redundant sequences are removed, the number of unique DH segment sequences in this illustrative embodiment of the library is 278. These sequences are enumerated in Table 18.

TABLE 18 DH Gene Segments Used in the Presently Exemplified Library* DH Segment SEQ ID DH Segment SEQ ID Designation¹ Peptide NO: Designation Peptide NO: IGHD1-26_1-1 ATT (a) IGHD3-10_2- YYGSG (h) 20 IGHD1-26_1-2 GAT (a) IGHD3-10_2- YYYGS (h) 21 IGHD1-26_1-3 GIV (a) IGHD3-10_2- GSGSYY (h) 22 IGHD1-26_1-4 IVG (a) IGHD3-10_2- SGSYYN (h) 23 IGHD1-26_1-5 VGA (a) IGHD3-10_2- YGSGSY (h) 24 IGHD1-26_1-6 GATT (a) IGHD3-10_2- YYGSGS (h) 25 IGHD1-26_1-7 GIVG (a) IGHD3-10_2- YYYGSG (h) 26 IGHD1-26_1-8 IVGA (a) IGHD3-10_2- GSGSYYN (h) 27 IGHD1-26_1-9 VGAT (a) IGHD3-10_2- YGSGSYY (h) 28 IGHD1-26_1-10 GIVGA (a) IGHD3-10_2- YYGSGSY (h) 29 IGHD1-26_1-11 IVGAT (a) IGHD3-10_2- YYYGSGS (h) 30 IGHD1-26_1-12 VGATT (a) IGHD3-10_2- YGSGSYYN (h) 31 IGHD1-26_1-13 GIVGAT (a) IGHD3-10_2- YYGSGSYY (h) 32 IGHD1-26_1-14 IVGATT (a) IGHD3-10_2- YYYGSGSY (h) 33 IGHD1-26_1-15 GIVGATT 13 IGHD3-10_2- YYGSGSYYN (h) 34 IGHD1-26_3-1 YSG (b) IGHD3-10_2- YYYGSGSYY (h) 35 IGHD1-26_3-2 YSGS (b) IGHD3-10_2- YYYGSGSYYN  2 36 IGHD1-26_3-3 YSGSY (b) IGHD3-10_3-1 GVI (i) IGHD1-26_3-4 YSGSYY 14 IGHD3-10_3-2 ITM (i) IGHD2-02_2-1 CSSTSC (c) IGHD3-10_3-3 MVR (i) IGHD2-02_2-2 CSSTSCY (c) IGHD3-10_3-4 RGV (i) IGHD2-02_2-3 YCSSTSC (c) IGHD3-10_3-5 TMV (i) IGHD2-02_2-4 CSSTSCYT (c) IGHD3-10_3-6 VII (i) IGHD2-02_2-5 GYCSSTSC (c) IGHD3-10_3-7 VRG (i) IGHD2-02_2-6 YCSSTSCY (c) IGHD3-10_3-8 GVII (i) IGHD2-02_2-7 GYCSSTSCY (c) IGHD3-10_3-9 ITMV (i) IGHD2-02_2-8 YCSSTSCYT (c) IGHD3-10_3- MVRG (i) 10 IGHD2-02_2-9 GYCSSTSCYT 10 IGHD3-10_3- RGVI (i) 11 IGHD2-02_3-1 AAM (d) IGHD3-10_3- TMVR (i) 12 IGHD2-02_3-2 DIV (d) IGHD3-10_3- VRGV (i) 13 IGHD2-02_3-3 IVV (d) IGHD3-10_3- ITMVR (i) 14 IGHD2-02_3-4 PAA (d) IGHD3-10_3- MVRGV (i) 15 IGHD2-02_3-5 VPA (d) IGHD3-10_3- RVGII (i) 16 IGHD2-02_3-6 VVP (d) IGHD3-10_3- TMVRG (i) 17 IGHD2-02_3-7 VVV (d) IGHD3-10_3- VRGVI (i) 18 IGHD2-02_3-8 DIVV (d) IGHD3-10_3- ITMVRG (i) 19 IGHD2-02_3-9 IVVV (d) IGHD3-10_3- MVRGVI (i) 20 IGHD2-02_3-10 PAAM (d) IGHD3-10_3- TMVRGV (i) 21 IGHD2-02_3-11 VPAA (d) IGHD3-10_3- VRGVII (i) 22 IGHD2-02_3-12 VVPA (d) IGHD3-10_3- ITMVRGV (i) 23 IGHD2-02_3-13 VVVP (d) IGHD3-10_3- MVRGVII (i) 24 IGHD2-02_3-14 DIVVV (d) IGHD3-10_3- TMVRGVI (1) 25 IGHD2-02_3-15 IVVVP (d) IGHD3-10_3- ITMVRGVI (i) 26 IGHD2-02_3-16 VPAAM (d) IGHD3-10_3- TMVRGVII (i) 27 IGHD2-02_3-17 VVPAA (d) IGHD3-10_3- ITMVRGVII  3 28 IGHD2-02_3-18 VVVPA (d) IGHD3-22_2-1 DSS (j) IGHD2-02_3-19 DIVVVP (d) IGHD3-22_2-2 GYY (j) IGHD2-02_3-20 IVVVPA (d) IGHD3-22_2-3 SGY (j) IGHD2-02_3-21 VVPAAM (d) IGHD3-22_2-4 SSG (j) IGHD2-02_3-22 VVVPAA (d) IGHD3-22_2-5 YDS (j) IGHD2-02_3-23 DIVVVPA (d) IGHD3-22_2-6 YYD (j) IGHD2-02_3-24 IVVVPAA (d) IGHD3-22_2-7 DSSG (j) IGHD2-02_3-25 VVVPAAM (d) IGHD3-22_2-8 GYYY (j) IGHD2-02_3-26 DIVVVPAA (d) IGHD3-22_2-9 SGYY (j) IGHD2-02_3-27 IVVVPAAM (d) IGHD3-22_2- SSGY (j) 10 IGHD2-02_3-28 DIVVVPAAM 11 IGHD3-22_2- YDSS (j) 11 IGHD2-15_2-1 CSGGSC (e) IGHD3-22_2- YYDS (j) 12 IGHD2-15_2-2 CSGGSCY (e) IGHD3-22_2- YYYD (j) 13 IGHD2-15_2-3 YCSGGSC (e) IGHD3-22_2- DSSGY (j) 14 IGHD2-15_2-4 CSGGSCYS (e) IGHD3-22_2- SGYYY (j) 15 IGHD2-15_2-5 GYCSGGSC (e) IGHD3-22_2- SSGYY (j) 16 IGHD2-15_2-6 YCSGGSCY (e) IGHD3-22_2- YDSSG (j) 17 IGHD2-15_2-7 GYCSGGSCY (e) IGHD3-22_2- YYDSS (j) 18 IGHD2-15_2-8 YCSGGSCYS (e) IGHD3-22_2- YYYDS (j) 19 IGHD2-15_2-9 GYCSGGSCYS 16 IGHD3-22_2- DSSGYY (j) 20 IGHD3-03_3-1 FGV (f) IGHD3-22_2- SSGYYY (j) 21 IGHD3-03_3-2 GVV (f) IGHD3-22_2- YDSSGY (j) 22 IGHD3-03_3-3 IFG (f) IGHD3-22_2- YYDSSG (j) 23 IGHD3-03_3-4 ITI (f) IGHD3-22_2- YYYDSS (j) 24 IGHD3-03_3-5 TIF (f) IGHD3-22_2- DSSGYYY (j) 25 IGHD3-03_3-6 VVI (f) IGHD3-22_2- YDSSGYY (j) 26 IGHD3-03_3-7 FGVV (f) IGHD3-22_2- YYDSSGY (j) 27 IGHD3-03_3-8 GVVI (f) IGHD3-22_2- YYYDSSG (j) 28 IGHD3-03_3-9 IFGV (f) IGHD3-22_2- YDSSGYYY (j) 29 IGHD3-03_3-10 ITIF (f) IGHD3-22_2- YYDSSGYY (j) 30 IGHD3-03_3-11 TIFG (f) IGHD3-22_2- YYYDSSGY (j) 31 IGHD3-03_3-12 VVII (f) IGHD3-22_2- YYDSSGYYY (j) 32 IGHD3-03_3-13 FGVVI (f) IGHD3-22 2- YYYDSSGYY (j) 33 IGHD3-03_3-14 GVVII (f) IGHD3-22_2- YYYDSSGYYY (j) 34 IGHD3-03_3-15 IFGVV (f) IGHD4-17_2-1 DYG (k) IGHD3-03_3-16 ITIFG (f) IGHD4-17_2-2 GDY (k) IGHD3-03_3-17 TIFGV (f) IGHD4-17_2-3 YGD (k) IGHD3-03_3-18 FGVVII (f) IGHD4-17_2-4 DYGD (k) IGHD3-03_3-19 IFGVVI (f) IGHD4-17_2-5 YGDY (k) IGHD3-03_3-20 ITIFGV (f) IGHD4-17_2-6 DYGDY 12 IGHD3-03_3-21 TIFGVV (f) IGHD5-5_3-1 SYG (l) IGHD3-03_3-22 IFGVVII (f) IGHD5-5_3-2 YGY (l) IGHD3-03_3-23 ITIFGVV (f) IGHD5-5_3-3 YSY (l) IGHD3-03_3-24 TIFGVVI (f) IGHD5-5_3-4 GYSY (l) IGHD3-03_3-25 ITIFGVVI (f) IGHD5-5_3-5 SYGY (l) IGHD3-03_3-26 TIFGVVII (f) IGHD5-5_3-6 YSYG (l) IGHD3-03_3-27 ITIFGVVII  9 IGHD5-5_3-7 GYSYG (l) IGHD3-10_1-1 ELL (g) IGHD5-5_3-8 YSYGY (l) IGHD3-10_1-2 FGE (g) IGHD5-5_3-9 GYSYGY 15 IGHD3-10_1-3 GEL (g) IGHD6-13_1-1 SSS (m) IGHD3-10_1-4 LLW (g) IGHD6-13_1-2 SSW (m) IGHD3-10_1-5 LWF (g) IGHD6-13_1-3 SWY (m) IGHD3-10_1-6 VLL (g) IGHD6-13_1-4 SSSW (m) IGHD3-10_1-7 WFG (g) IGHD6-13_1-5 SSWY (m) IGHD3-10_1-8 FGEL (g) IGHD6-13_1-6 YSSS (m) IGHD3-10_1-9 GELL (g) IGHD6-13_1-7 GYSSS (m) IGHD3-10_1-10 LLWF (g) IGHD6-13_1-8 SSSWY (m) IGHD3-10_1-11 LWFG (g) IGHD6-13_1-9 YSSSW (m) IGHD3-10_1-12 VLLW (g) IGHD6-13_1- GYSSSW (m) 10 IGHD3-10_1-13 WFGE (g) IGHD6-13_1- YSSSWY (m) 11 IGHD3-10_1-14 FGELL (g) IGHD6-13_1- GYSSSWY  7 12 IGHD3-10_1-15 LLWFG (g) IGHD6-19_1-1 GWY (n) IGHD3-10_1-16 LWFGE (g) IGHD6-19_1-2 GYS (n) IGHD3-10_1-17 VLLWF (g) IGHD6-19_1-3 SGW (n) IGHD3-10_1-18 WFGEL (g) IGHD6-19_1-4 YSS (n) IGHD3-10_1-19 LLWFGE (g) IGHD6-19_1-5 GYSS (n) IGHD3-10_1-20 LWFGEL (g) IGHD6-19_1-6 SGWY (n) IGHD3-10_1-21 VLLWFG (g) IGHD6-19_1-7 SSGW (n) IGHD3-10_1-22 WFGELL (g) IGHD6-19_1-8 YSSG (n) IGHD3-10_1-23 LLWFGEL (g) IGHD6-19_1-9 GYSSG (n) IGHD3-10_1-24 LWFGELL (g) IGHD6-19_1- SSGWY (n) 10 IGHD3-10_1-25 VLLWFGE (g) IGHD6-19_1- YSSGW (n) 11 IGHD3-10_1-26 LLWFGELL (g) IGHD6-19_1- GYSSGW (n) 12 IGHD3-10_1-27 VLLWFGEL (g) IGHD6-19_1- YSSGWY (n) 13 IGHD3-10_1-28 VLLWFGELL  1 IGHD6-19_1- GYSSGWY  5 14 IGHD3-10_2-1 GSG (h) IGHD6-19_2-1 AVA (o) IGHD3-10_2-2 GSY (h) IGHD6-19_2-2 GIA (o) IGHD3-10_2-3 SGS (h) IGHD6-19_2-3 IAV (o) IGHD3-10_2-4 SYY (h) IGHD6-19_2-4 VAG (o) IGHD3-10_2-5 YGS (h) IGHD6-19_2-5 AVAG (o) IGHD3-10_2-6 YYG (h) IGHD6-19_2-6 GIAV (o) IGHD3-10_2-7 YYN (h) IGHD6-19_2-7 IAVA (o) IGHD3-10_2-8 YYY (h) IGHD6-19_2-8 GIAVA (o) IGHD3-10_2-9 GSGS (h) IGHD6-19_2-9 IAVAG (o) IGHD3-10_2-10 GSYY (h) IGHD6-19_2- GIAVAG  6 10 IGHD3-10_2-11 SGSY (h) IGHD6-13_2-1 AAA (p) IGHD3-10_2-12 SYYN (h) IGHD6-13_2-2 AAG (p) IGHD3-10_2-13 YGSG (h) IGHD6-13_2-3 IAA (p) IGHD3-10_2-14 YYGS (h) IGHD6-13_2-4 AAAG (p) IGHD3-10_2-15 YYYG (h) IGHD6-13_2-5 GIAA (p) IGHD3-10_2-16 GSGSY (h) IGHD6-13_2-6 IAAA (p) IGHD3-10_2-17 GSYYN (h) IGHD6-13_2-7 GIAAA (p) IGHD3-10_2-18 SGSYY (h) IGHD6-13_2-8 IAAAG (p) IGHD3-10_2-19 YGSGS (h) IGHD6-13_2-9 GIAAAG 8 ¹The sequence designation is formatted as follows: (IGHD Gene Name)_(Reading Frame)-(Variant Number) *Note that the origin of certain variants is rendered somewhat arbitrary when redundant segments are deleted from the library (i.e., certain segments may have their origins with more than one parent, including the one specified in the table). (a) derived from SEQ ID NO: 13; (b) derived from SEQ ID NO: 14; (c) derived from SEQ ID NO: 10; (d) derivedfrom SEQ ID NO: 11; (e) derived from SEQ ID NO: 16; (f) derived from SEQ ID NO: 9; (g) derived from SEQ ID NO: 1; (h) derived from SEQ ID NO: 2; (i) derived from SEQ ID NO: 3; (j) derived from SEQ ID NO: 4; (k) derived from SEQ ID NO: 12; (l) derived from SEQ ID NO: 15; (m) derived from SEQ ID NO: 7; (n) derived from SEQ ID NO: 5; (o) derived from SEQ ID NO: 6; (p) derived from SEQ ID NO: 8.

Table 19 shows the length distribution of the 278 DH segments selected according to the methods described above.

TABLE 19 Length Distributions of DH Segments Selected for Inclusion in the Exemplary Library Number of DH Size Occurrences 3 78 4 64 5 50 6 38 7 27 8 20 9 12 10 4

As specified above, based on the CDRH3 numbering system defined in this application, IGHD-derived amino acids (i.e., DH segments) are numbered beginning with position 97, followed by positions 97A, 97B, etc. In the currently exemplified embodiment of the library, the shortest DH segment has three amino acids: 97, 97A and 97B, while the longest DH segment has 10 amino acids: 97, 97A, 97B, 97C, 97D, 97E, 97F, 97G, 97H and 97I.

Example 5.2 Selection of the H3-JH Segments

There are six human germline IGHJ genes. During in vivo assembly of antibody genes, these segments are progressively deleted at their 5′ end. In this exemplary embodiment of the library, IGHJ gene segments with no deletions, or with 1, 2, 3, 4, 5, 6, or 7 deletions (at the amino acid level), yielding JH segments as short as 13 amino acids, were included (Table 20). Other embodiments of the invention, in which the IGHJ gene segments are progressively deleted (at their 5′/N-terminal end) to yield 15, 14, 12, or 11 amino acids are also contemplated.

TABLE 20 IGHJ Gene Segments Selected for use in the Exemplary Library IGHJ SEQ ID Segment [H3-JH]-[FRM4]¹ NO: H3-JH SEQ ID NO: JH1 parent or    AEYFQHWGQGTLVTVSS 253    AEYFQH 17 JH1_1 JH1_2     EYFQHWGQGTLVTVSS (a)     EYFQH (g) JH1_3      YFQHWGQGTLVTVSS (a)      YFQH (g) JH1_4       FQHWGQGTLVTVSS (a)       FQH (g) JH1_5        QHWGQGTLVTVSS (a)        QH (g) JH2 parent or    YWYFDLWGRGTLVTVSS 254    YWYFDL 18 JH2_1 JH2_2     WYFDLWGRGTLVTVSS (b)     WYFDL (h) JH2_3      YFDLWGRGTLVTVSS (b)      YFDL (h) JH2_4       FDLWGRGTLVTVSS (b)       FDL (h) JH2_5        DLWGRGTLVTVSS (b)        DL (h) JH3 parent or      AFDVWGQGTMVTVSS 255      AFDV 19 JH3_1 JH3_2       FDVWGQGTMVTVSS (c)       FDV (i) JH3_3        DVWGQGTMVTVSS (c)        DV (i) JH4 parent or      YFDYWGQGTLVTVSS 256      YFDY 20 JH4_1 JH4_2       FDYWGQGTLVTVSS (d)       FDY (j) JH4_3        DYWGQGTLVTVSS (d)        DY (j) JH5 parent or     NWFDSWGQGTLVTVSS 257     NWFDS 21 JH5_1 JH5_2      WFDSWGQGTLVTVSS (e)      WFDS (k) JH5_3       FDSWGQGTLVTVSS (e)       FDS (k) JH5_4        DSWGQGTLVTVSS (e)        DS (k) JH6 parent or YYYYYGMDVWGQGTTVTVSS 258 YYYYYGMDV 22 JH6_1 JH6_2  YYYYGMDVWGQGTTVTVSS (f)  YYYYGMDV (l) JH6_3   YYYGMDVWGQGTTVTVSS (f)   YYYGMDV (l) JH6_4    YYGMDVWGQGTTVTVSS (f)    YYGMDV (l) JH6_5     YGMDVWGQGTTVTVSS (f)     YGMDV (l) JH6_6      GMDVWGQGTTVTVSS (f)      GMDV (l) JH6_7       MDVWGQGTTVTVSS (f)       MDV (l) JH6_8        DVWGQGTTVTVSS (f)        DV (l) ¹H3-JH is defined as the portion of the IGHJ segment included within the Kabat definition of CDRH3; FRM4 is defined as the portion of the IGHJ segment encoding framework region four. (a) derived from SEQ ID NO: 253; (b) derived from SEQ ID NO: 254; (c) derived from SEQ ID NO: 255; (d) derived from SEQ ID NO: 256; (e) derived from SEQ ID NO: 257; (f) derived from SEQ ID NO: 258; (g) derived from SEQ ID NOs: 17 and 253; (h) derived from SEQ ID NOs: 18 and 254; (i) derived from SEQ ID NOs: 19 and 255; (j) derived from SEQ ID NOs: 20 and 256; (k) derived from SEQ ID NOs: 21 and 257; (l) derived from SEQ ID NOs: 22 and 258.

According to the CDRH3 numbering system of this application, the contribution of, for example, JH6_(—)1 to CDRH3, would be designated by positions 99F, 99E, 99D, 99C, 99B, 99A, 100, 101 and 102 (Y, Y, Y, Y, Y, G, M, D and V, respectively). Similarly, the JH4_(—)3 sequence would contribute amino acid positions 101 and 102 (D and Y, respectively) to CDRH3. However, in all cases of the exemplified library, the JH segment will contribute amino acids 103 to 113 to the FRM4 region, in accordance with the standard Kabat numbering system for antibody variable regions (Kabat, op. cit. 1991). This may not be the case in other embodiments of the library.

Example 5.3 Selection of the N1 and N2 Segments

While the consideration of V-D-J recombination enhanced by mimicry of the naturally occurring process of progressive deletion (as exemplified above) can generate enormous diversity, the diversity of the CDRH3 sequences in vivo is further amplified by non-templated addition of a varying number of nucleotides at the V-D junction and the D-J junction.

N1 and N2 segments located at the V-D and D-J junctions, respectively, were identified in a sample containing about 2,700 antibody sequences (Jackson et al., J. Immunol. Methods, 2007, 324: 26) also analyzed by the SoDA method of Volpe et al., Bioinformatics, 2006, 22: 438-44; (both Jackson et al., and Volpe et al., are incorporated by reference in their entireties). Examination of these sequences revealed patterns in the length and composition of N1 and N2. For the construction of the currently exemplified CDRH3 library, specific short amino acid sequences were derived from the above analysis and used to generate a number of N1 and N2 segments that were incorporated into the CDRH3 design, using the synthetic scheme described herein.

As described in the Detailed Description, certain embodiments of the invention include N1 and N2 segments with rationally designed length and composition, informed by statistical biases in these parameters that are found by comparing naturally occurring N1 and N2 segments in human antibodies. According to data compiled from human databases (see, e.g., Jackson et al., J. Immunol. Methods, 2007, 324: 26, incorporated by reference in its entirety), there are an average of about 3.02 amino acid insertions for N1 and about 2.4 amino acid insertions for N2, not taking into account insertions of two nucleotides or less. FIG. 2 shows the length distributions of the N1 and N2 regions in human antibodies. In this exemplary embodiment of the invention, N1 and N2 were fixed to a length of 0, 1, 2, or 3 amino acids. The naturally occurring composition of these sequences in human antibodies was used as a guide for the inclusion of different amino acid residues.

The naturally occurring composition of single amino acid, two amino acids, and three amino acids N1 additions is defined in Table 21, and the naturally occurring composition of the corresponding N2 additions is defined in Table 22. The most frequently occurring duplets in the N1 and N2 set are compiled in Table 23.

TABLE 21 Composition of Naturally Occurring 1, 2, and 3 Amino Acid N1 Additions* Posi- Number of Posi- Number of Posi- Number of tion 1 Occurrences tion 2 Occurrences tion 3 Occurrences R 251 G 97 G 101 G 249 P 67 R 66 P 173 R 67 P 47 L 130 S 42 S 47 S 117 L 39 L 38 A 84 V 33 A 33 V 62 E 24 V 28 K 61 A 21 T 27 I 55 D 18 E 24 Q 51 I 18 D 22 T 51 T 18 K 18 D 50 K 16 F 14 E 49 Y 16 I 13 F 3 H 13 W 13 H 32 F 12 N 10 N 30 Q 11 Y 10 W 28 N 5 H 8 Y 21 W 5 Q 5 M 16 C 4 C 3 C 3 M 4 M 3 1546 530 530 *Defined as the sequence C-terminal to “CARX”, or equivalent, of VH, wherein “X” is the “tail” (e.g., D, E, G, or no amino acid residue).

TABLE 22 Composition of Naturally Occurring 1, 2, and 3 Amino Acid N2 Additions* Number of Position Number of Position Number of Position 1 Occurrences 2 Occurrences 3 Occurrences G 242 G 244 G 156 P 219 P 138 P 79 R 180 R 86 S 54 L 132 S 85 R 51 S 123 T 77 L 49 A 97 L 74 A 41 T 78 A 69 T 31 V 75 V 46 V 29 E 57 E 41 D 23 D 56 Y 38 E 23 F 54 D 36 W 23 H 54 K 30 Q 19 Q 53 F 29 F 17 I 49 W 27 Y 17 N 45 H 24 H 16 Y 40 I 23 I 11 K 35 Q 23 K 11 W 29 N 21 N 8 M 20 M 8 C 6 C 6 C 5 M 6 1644 1124 670 *Defined as the sequence C-terminal to the D segment but not encoded by IGHJ genes.

TABLE 23 Top Twenty-Five Naturally Occurring N1 and N2 Duplets Number of Cumulative Individual Sequence Occurrences Frequency Frequency GG 17 0.037 0.037 PG 15 0.070 0.033 RG 15 0.103 0.033 PP 13 0.132 0.029 GP 12 0.158 0.026 GL 11 0.182 0.024 PT 10 0.204 0.022 TG 10 0.226 0.022 GV 9 0.246 0.020 RR 9 0.266 0.020 SG 8 0.284 0.018 RP 7 0.299 0.015 IG 6 0.312 0.013 GS 6 0.325 0.013 SR 6 0.338 0.013 PA 6 0.352 0.013 LP 6 0.365 0.013 VG 6 0.378 0.013 KG 6 0.389 0.011 GW 5 0.400 0.011 FP 5 0.411 0.011 LG 5 0.422 0.011 RS 5 0.433 0.011 TP 5 0.444 0.011 EG 5 0.455 0.011

Example 5.3.1 Selection of the N1 Segments

Analysis of the identified N1 segments, located at the junction between V and D, revealed that the eight most frequently occurring amino acid residues were G, R, S, P, L, A, T and V (Table 21). The number of amino acid additions in the N1 segment was frequently none, one, two, or three (FIG. 2). The addition of four or more amino acids was relatively rare. Therefore, in the currently exemplified embodiment of the library, the N1 segments were designed to include zero, one, two or three amino acids. However, in other embodiments, N1 segments of four, five, or more amino acids may also be utilized. G and P were always among the most commonly occurring amino acid residues in the N1 regions. Thus, in the present exemplary embodiment of the library, the N1 segments that are dipeptides are of the form GX, XG, PX, or XP, where X is any of the eight most commonly occurring amino acids listed above. Due to the fact that G residues were observed more frequently than P residues, the tripeptide members of the exemplary N1 library have the form GXG, GGX, or XGG, where X is, again, one of the eight most frequently occurring amino acid residues listed above. The resulting set of N1 sequences used in the present exemplary embodiment of the library, include the “zero” addition amounts to 59 sequences, which are listed in Table 24.

TABLE 24 N1 Sequences Selected for Inclusion in the Exemplary Library Segment Type Sequences Number “Zero” (no addition) V segment joins 1 directly to D segment Monomers G, P, R, A, S, L, T, V 8 Dimers GG, GP, GR, GA, GS, GL, GT, GV, PG, 28 RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP Trimers GGG, GPG, GRG, GAG, GSG, GLG, GTG, 22 GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV

In accordance with the CDRH3 numbering system of the application, the sequences enumerated in Table 24 contribute the following positions to CDRH3: the monomers contribute position 96, the dimers to 96 and 96A, and the trimers to 96, 96A and 96B. In alternative embodiments, where tetramers and longer segments could be included among the N1 sequences, the corresponding numbers would go on to include 96C, and so on.

Example 5.3.2 Selection of the N2 Segments

Similarly, analysis of the identified N2 segments, located at the junction between D and J, revealed that the eight most frequently occurring amino acid residues were also G, R, S, P, L, A, T and V (Table 22). The number of amino acid additions in the N2 segment was also frequently none, one, two, or three (FIG. 2). For the design of the N2 segments in the exemplary library, an expanded set of sequences was utilized. Specifically, the sequences in Table 25 were used, in addition to the 59 sequences enumerated in Table 24, for N1.

TABLE 25 Extra Sequences in N2 Additions Segment Number Number Type Sequence New Total Monomers D, E, F, H, I, K, M, Q, W, Y 10 18 Dimers AR, AS, AT, AY, DL, DT, EA, 54 82 EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS Trimers AAE, AYH, DTL, EKR, ISR, NTP, 18 40 PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT

The presently exemplified embodiment of the library, therefore, contains 141 total N2 sequences, including the “zero” state. One of ordinary skill in the art will readily recognize that these 141 sequences may also be used in the N1 region, and that such embodiments are within the scope of the invention. In addition, the length and compositional diversity of the N1 and N2 sequences can be further increased by utilizing amino acids that occur less frequently than G, R, S, P, L, A, T and V, in the N1 and N2 regions of naturally occurring antibodies, and including N1 and N2 segments of four, five, or more amino acids in the library. Tables 21 to 23 and FIG. 2 provides information about the composition and length of the N1 and N2 sequences in naturally occurring antibodies that is useful for the design of additional N1 and N2 regions which mimic the natural composition and length.

In accordance with the CDRH3 numbering system of the application, N2 sequences will begin at position 98 (when present) and extend to 98A (dimers) and 98B (trimers). Alternative embodiments may occupy positions 98C, 98D, and so on.

Example 5.4 A CDRH3 Library

When the “tail” (i.e., G/D/E/-) is considered, the CDRH3 in the exemplified library may be represented by the general formula:

[G/D/E/-]-[N1]-[DH]-[N2]-[H3-JH]

In the currently exemplified, non-limiting, embodiment of the library, [G/D/E/-] represents each of the four possible terminal amino acid “tails”; N1 can be any of the 59 sequences in Table 24; DH can be any of the 278 sequences in Table 18; N2 can be any of the 141 sequences in Tables 24 and 25; and H3-JH can be any of the 28 H3-JH sequences in Table 20. The total theoretical diversity or repertoire size of this CDRH3 library is obtained by multiplying the variations at each of the components, i.e., 4×59×278×141×28=2.59×10⁸.

However, as described in the previous examples, redundancies may be eliminated from the library. In the presently exemplified embodiment, the tail and N1 segments were combined, and redundancies were removed from the library. For example, considering the VH chassis, tail, and N1 regions, the sequence [VH_Chassis]-[G] may be obtained in two different ways: [VH_Chassis]+[G]+[nothing] or [VH_Chassis]+[nothing]+[G]. Removal of redundant sequences resulted in a total of 212 unique [G/D/E/-]-[N1] segments out of the 236 possible combinations (i.e., 4 tails×59 N1). Therefore, the actual diversity of the presently exemplified CDRH3 library is 212×278×141×28=2.11×10⁸. FIG. 23 depicts the frequency of occurrence of different CDRH3 lengths in this library, versus the preimmune repertoire of Lee et al.

Table 26 further illustrates specific exemplary sequences from the CDRH3 library described above, using the CDRH3 numbering system of the present application. In instances where a position is not used, the hyphen symbol (−) is included in the table instead.

TABLE 26 Examples of Designed CDRH3 Sequences According to the Library Exemplified in Examples 1 to 5 [Tail] [N1] [DH] [N2] [H3-JH] CDRH3 95 96 96A 96B 97 97A 97B 97C 97D 97E 97F 97G 97H 97I 98 98A 98B 99E 99D 99C 99B 99A 99 100 101 102 Length No. 1 G — — — Y Y Y — — — — — — — — — — — — — — — — — D V  6 No. 2 D G — — G Y C S G G S C Y S Y — — — — — — — F Q H 16 No. 3 E R — — I T I F G V — — — — G G — — — — — — Y F D Y 14 No. 4 — P P — V L L W F G E L L — D — — — — — — — — — D L 14 No. 5 G G S G Y Y Y G S G S Y Y N P — — — — — A E Y F Q H 21 No. 6 D — — — R G V I I — — — — — M — — Y Y Y Y Y G M D V 16 No. 7 E S G — Y Y Y D S S G Y Y Y T G L — — — — W Y F D L 21 No. 8 — S — — D Y G D Y — — — — — S I — — — — — — — F D I 11 No. 9 — P G — W F G — — — — — — — P S — — — — Y Y G M D V 13 No. 10 — — — — C S G G S C — — — — A Y — — — — — N W F D P 13 Sequence Identifiers No. 1 (SEQ ID NO: 542); No. 2 (SEQ ID NO: 543); No. 3 (SEQ ID NO: 544); No. 4 (SEQ ID NO: 545); No. 5 (SEQ ID NO: 546); No. 6 (SEQ ID NO: 547); No. 7 (SEQ ID NO: 548); No. 8 (SEQ ID NO: 549); No. 9 (SEQ ID NO: 550); No. 10 (SEQ ID NO: 551).

Example 6 Design of VKCDR3 Libraries

This example describes the design of a number of exemplary VKCDR3 libraries. As specified in the Detailed Description, the actual version(s) of the VKCDR3 library made or used in particular embodiments of the invention will depend on the objectives for the use of the library. In this example the Kabat numbering system for light chain variable regions was used.

In order to facilitate examination of patterns of occurrence, human kappa light chain sequences were obtained from the publicly available NCBI database (Appendix A). As for the heavy chain sequences (Example 2), each of the sequences obtained from the publicly available database was assigned to its closest germline gene, on the basis of sequence identity. The amino acid compositions at each position were then determined within each kappa light chain subset.

Example 6.1 A Minimalist VKCDR3 Library

This example describes the design of a “minimalist” VKCDR3 library, wherein the VKCDR3 repertoire is restricted to a length of nine residues. Examination of the VKCDR3 lengths of human sequences shows that a dominant proportion (over 70%) has nine amino acids within the Kabat definition of CDRL3: positions 89 through 97. Thus, the currently exemplified minimalist design considers only VKCDR3 of length nine. Examination of human kappa light chain sequences shows that there are not strong biases in the usage of IGKJ genes; there are five such IKJ genes in humans. Table 27 depicts IGKJ gene usage amongst three data sets, namely Juul et al. (Clin. Exp. Immunol., 1997, 109: 194, incorporated by reference in its entirety), Klein and Zachau (Eur. J. Immunol., 1993, 23: 3248, incorporated by reference in its entirety), and the kappa light chain data set provided in Appendix A (labeled LUA).

TABLE 27 IGKJ Gene Usage in Various Data Sets Gene Klein Juul LUA IGKJ1 35.0% 29.0% 29.3% IGKJ2 25.0% 23.0% 24.1% IGKJ3 7.0% 8.0% 12.1% IGKJ4 26.0% 24.0% 26.5% IGKJ5 6.0% 18.0% 8.0%

Thus, a simple combinatorial of “M” VK chassis and the 5 IGKJ genes would generate a library of size M×5. In the Kabat numbering system, for VKCDR3 of length nine, amino acid number 96 is the first encoded by the IGKJ gene. Examination of the amino acid occupying this position in human sequences showed that the seven most common residues are L, Y, R, W, F, P, and I, cumulatively accounting for about 85% of the residues found in position 96. The remaining 13 amino acids account for the other 15%. The occurrence of all 20 amino acids at position 96 is presented in Table 28.

TABLE 28 Occurrence of 20 Amino Acid Residues at Position 96 in Human VK Data Set Type Number Percent Cumulative L 333 22.3 22.3 Y 235 15.8 38.1 R 222 14.9 52.9 W 157 10.5 63.5 F 148 9.9 73.4 I 96 6.4 79.8 P 90 6.0 85.9 Q 53 3.6 89.4 N 39 2.6 92.0 H 31 2.1 94.1 V 21 1.4 95.5 G 20 1.3 96.8 C 14 0.9 97.8 K 7 0.5 98.3 S 6 0.4 98.7 A 5 0.3 99.0 D 5 0.3 99.3 E 5 0.3 99.7 T 5 0.3 100.0 M 0 0.0 100.0

To determine the origins of the seven residues most commonly found in position 96, known human IGKJ amino acid sequences were examined (Table 29).

TABLE 29 Known Human IGKJ Amino Acid Sequences Gene Sequence SEQ ID NO: IGKJ1 WTFGQGTKVEIK 552 IGKJ2 YTFGQGTKLEIK 553 IGKJ3 FTFGPGTKVDIK 554 IGKJ4 LTFGGGTKVEIK 555 IGKJ5 ITFGQGTRLEIK 556

Without being bound by theory, five of the seven most commonly occurring amino acids found in position 96 of rearranged human sequences appear to originate from the first amino acid encoded by each of the five human IGKJ genes, namely, W, Y, F, L, and I.

Less evident were the origins of the P and R residues. Without being bound by theory, most of the human IGKV gene nucleotide sequences end with the sequence CC, which occurs after (i.e., 3′ to) the end of the last full codon (e.g., that encodes the C-terminal residue shown in Table 11). Therefore, regardless of which nucleotide is placed after this sequence (i.e., CCX, where X may be any nucleotide) the codon will encode a proline (P) residue. Thus, when the IGKJ gene undergoes progressive deletion (just as in the IGHJ of the heavy chain; see Example 5), the first full amino acid is lost and, if no deletions have occurred in the IGKV gene, a P residue will result.

To determine the origin of the arginine residue at position 96, the origin of IGKJ genes in rearranged kappa light chain sequences containing R at position 96 were analyzed. The analysis indicated that R occurred most frequently at position 96 when the IGKJ gene was IGKJ1. The germline W (position 1; Table 29) for IGKJ1 is encoded by TGG. Without being bound by theory, a single nucleotide change of T to C (yielding CGG) or A (yielding AGG) will, therefore, result in codons encoding Arg (R). A change to G (yielding GGG) results in a codon encoding Gly (G). R occurs about ten times more often at position 96 in human sequences than G (when the IGKJ gene is IGKJ1), and it is encoded by CGG more often than AGG. Therefore, without being bound by theory, C may originate from one of the aforementioned two Cs at the end of IGKV gene. However, regardless of the mechanism(s) of occurrence, R and P are among the most frequently observed amino acid types at position 96, when the length of VKCDR3 is 9. Therefore, a minimalist VKCDR3 library may be represented by the following amino acid sequence:

[VK_Chassis]-[L3-VK]-[F/L/I/R/W/Y/P]-[TFGGGTKVEIK]

In this sequence, VK_Chassis represents any selected VK chassis (for non-limiting examples, see Table 11), specifically Kabat residues 1 to 88 encoded by the IGKV gene. L3-VK represents the portion of the VKCDR3 encoded by the chosen IGKV gene (in this embodiment, residues 89-95). F/L/J/R/W/Y/P represents any one of amino residues F, L, I, R, W, Y, or P. In this exemplary representation, IKJ4 (minus the first residue) has been depicted. Without being bound by theory, apart from IGKJ4 being among the most commonly used IGKJ genes in humans, the GGG amino acid sequence is expected to lead to larger conformational flexibility than any of the alternative IGKJ genes, which contain a GXG amino acid sequence, where X is an amino acid other than G. In some embodiments, it may be advantageous to produce a minimalist pre-immune repertoire with a higher degree of conformational flexibility. Considering the ten VK chassis depicted in Table 11, one implementation of the minimalist VKCDR3 library would have 70 members resulting from the combination of 10 VK chassis by 7 junction (position 96) options and one IGKJ-derived sequence (e.g., IGKJ4). Although this embodiment of the library has been depicted using IGKJ4, it is possible to design a minimalist VKCDR3 library using one of the other four IGKJ sequences. For example, another embodiment of the library may have 350 members (10 VK chassis by 7 junctions by 5 IGKJ genes).

One of ordinary skill in the art will readily recognize that one or more minimalist VKCDR3 libraries may be constructed using any of the IGKJ genes. Using the notation above, these minimalist VKCDR3 libraries may have sequences represented by, for example:

-   -   JK1: [VK_Chassis]-[L3-VK]-[F/L/I/R/W/Y/P]-[TFGQGTKVEIK];     -   JK2: [VK_Chassis]-[L3-VK]-[F/L/I/R/W/Y/P]-[TFGQGTKLEIK];     -   JK3: [VK_Chassis]-[L3-VK]-[F/L/I/R/W/Y/P]-[TFGPGTKVDIK]; and     -   JK4: [VK_Chassis]-[L3-VK]-[F/L/I/R/W/Y/P]-[TFGQGTRLEIK].

Example 6.2 A VKCDR3 Library of About 10⁵ Complexity

In this example, the nine residue VKCDR3 repertoire described in Example 6.1 is expanded to include VKCDR3 lengths of eight and ten residues. Moreover, while the previously enumerated VKCDR3 library included the VK chassis and portions of the IGKJ gene not contributing to VKCDR3, the presently exemplified version focuses only on residues comprising a portion of VKCDR3. This embodiment may be favored, for example, when recombination with a vector which already contains VK chassis sequences and constant region sequences is desired.

While the dominant length of VKCDR3 sequences in humans is nine amino acids, other lengths appear at measurable rates that cumulatively approach almost 30% of kappa light chain sequences. In particular, VKCDR3 of lengths 8 and 10 represent, respectively, about 8.5% and about 16% of sequences in representative samples (FIG. 3). Thus, a more complex VKCDR3 library includes CDR lengths of 8 to 10 amino acids; this library accounts for over 95% of the length distribution observed in typical collections of human VKCDR3 sequences. This library also enables the inclusion of additional variation outside of the junction between the VK and JK genes. The present example describes such a library. The library comprises 10 sub-libraries, each designed around one of the 10 exemplary VK chassis depicted in Table 11. Clearly, the approach exemplified here can be generalized to consider M different chassis, where M may be less than or more than 10.

To characterize the variability within the polypeptide segment occupying Kabat positions 89 to 95, human kappa light chain sequence collections derived from each of the ten germline sequences of Example 3 were aligned and compared separately (i.e., within the germline group). This analysis enabled us to discern the patterns of sequence variation at each individual position in each kappa light chain sequence, grouped by germline. The table below shows the results for sequences derived from IGKV1-39.

TABLE 30 Percent Occurrence of Amino Acid Types in IGKV1-39-Derived Sequences Amino Acid P89 P90 P91 P92 P93 P94 P95 A 0 0 1 0 0 4 1 C 0 0 0 0 0 0 0 D 0 0 1 1 3 0 0 E 0 1 0 0 0 0 0 F 0 0 0 5 0 2 0 G 0 0 2 1 2 0 0 H 1 1 0 4 0 0 0 I 0 0 1 0 4 5 1 K 0 0 0 1 2 0 0 L 3 0 0 1 1 3 7 M 0 0 0 0 0 1 0 N 0 0 3 2 6 2 0 P 0 0 0 0 0 4 85 Q 96 97 0 0 0 0 0 R 0 0 0 0 5 0 2 S 0 0 80 4 65 6 3 T 0 0 9 0 10 65 1 V 0 0 0 0 0 1 1 W 0 0 0 0 0 0 0 Y 0 0 2 80 0 3 0

For example, at position 89, two amino acids, Q and L, account for about 99% of the observed variability, and thus in the currently exemplified library (see below), only Q and L were included in position 89. In larger libraries, of course, additional, less frequently occurring amino acid types (e.g., H), may also be included.

Similarly, at position 93 there is more variation, with amino acid types S, T, N, R and I being among the most frequently occurring. The currently exemplified library thus aimed to include these five amino acids at position 93, although clearly others could be included in more diverse libraries. However, because this library was constructed via standard chemical oligonucleotide synthesis, one is bound by the limits of the genetic code, so that the actual amino acid set represented at position 93 of the exemplified library consists of S, T, N, R, P and H, with P and H replacing I (see exemplary 9 residue VKCDR3 in Table 32, below). This limitation may be overcome by using codon-based synthesis of oligonucleotides, as described in Example 6.3, below. A similar approach was followed at the other positions and for the other sequences: analysis of occurrences of amino acid type per position, choice from among most frequently occurring subset, followed by adjustment as dictated by the genetic code.

As indicated above, the library employs a practical and facile synthesis approach using standard oligonucleotide synthesis instrumentation and degenerate oligonucleotides. To facilitate description of the library, the IUPAC code for degenerate nucleotides, as given in Table 31, will be used.

TABLE 31 Degenerate Base Symbol Definition IUPAC Symbol Base Pair Composition A A (100%) C C (100%) G G (100%) T T (100%) R A (50%) G (50%) Y C (50%) T (50%) W A (50%) T (50%) S C (50%) G (50%) M A (50%) C (50%) K G (50%) T (50%) B C (33%) G (33%) T (33%) (*) D A (33%) G (33%) T (33%) H A (33%) C (33%) T (33%) V A (33%) C (33%) G (33%) N A (25%) C (25%) G (25%) T (25%) (*) 33% is short hand here for ⅓ (i.e., 33.3333 . . . %)

Using the VK1-39 chassis with VKCDR3 of length nine as an example, the VKCDR3 library may be represented by the following four oligonucleotides (left column in Table 32), with the corresponding amino acids encoded at each position of CDRL3 (Kabat numbering) provided in the columns on the right.

TABLE 32 Exemplary Oligonucleotides Encoding a VK1-39 CDR3 Library*** Oligonucleotide Sequence 89 90 91 92 93 94 95 95A 96 97 CWGSAAWCATHCMVTABTCCTTWCACT LQ EQ ST FSY HNPRST IST P — FY T (SEQ ID NO: 307) CWGSAAWCATHCMVTABTCCTMTCACT LQ EQ ST FSY HNPRST IST P — IL T (SEQ ID NO: 308) CWGSAAWCATHCMVTABTCCTWGGACT LQ EQ ST FSY HNPRST IST P — WR T (SEQ ID NO: 309) CWGSAAWCATHCMVTABTCCTCBTACT LQ EQ ST FSY HNPRST IST P PLR — T (SEQ ID NO: 310)

For example, the first codon (CWG) of the first nucleotide of Table 32, corresponding to Kabat position 89, represents 50% CTG and 50% CAG, which encode Leu (L) and Gln (O), respectively. Thus, the expressed polypeptide would be expected to have L and Q each about 50% of the time. Similarly, for Kabat position 95A of the fourth oligonucleotide, the codon CBT represents ⅓ each of CCT, CGT and CTT, corresponding in turn to ⅓ each of Pro (P), Leu (L) and Arg (R) upon translation. By multiplying the number of options available at each position of the peptide sequence, one can obtain the complexity, in peptide space, contributed by each oligonucleotide. For the VK1-39 example above, the numbers are 864 for the first three oligonucleotides and 1,296 for the fourth oligonucleotide. Thus, the oligonucleotides encoding VK1-39 CDR3s of length nine contribute 3,888 members to the library. However, as shown in Table 32, sequences with L or R at position 95A (when position 96 is empty) are identical to those with L or R at position 96 (and 95A empty). Therefore, the 3,888 number overestimates the LR contribution and the actual number of unique members is slightly lower, at 3,024. As depicted in Table 33, for the complete list of oligonucleotides that represent VKCDR3 of sizes 8, 9, and 10, for all 10 VK chassis, the overall complexity is about 1.3×10⁵ or 1.2×10⁵ unique sequences after correcting for over-counting of the LR contribution for the size 9 VKCDR3.

TABLE 33 Degenerate Oligonucleotides Encoding an Exemplary VKCDR3 Library SEQ CDR L3 Junction Degenerate ID Chassis Length Type (1) Oligonucleotide NO: 89 90 91 92 93 94 95 95A 96 97 VK1-5 8 1 CASCASTMCVRTRSTT 259 BQ HQ SY DGHNRS AGST FY — — FY T WCTWCACT VK1-5 8 2 CASCASTMCVRTRSTT 260 BQ HQ SY DGHNRS AGST FY — — IL T WCMTCACT VK1-5 8 3 CASCASTMCVRTRSTT 261 BQ HQ SY DGHNRS AGST FY — — WR T WCWGGACT VK1-5 8 4 CASCASTMCVRTRSTT 262 BQ HQ SY DGHNRS AGST FY PS — — T WCYCTACT VK1-5 9 1 CASCASTMCVRTRSTT 263 BQ HQ SY DGHNRS AGST FY PS — FY T WCYCTTWCACT VK1-5 9 2 CASCASTMCVRTRSTT 264 BQ HQ SY DGHNRS AGST FY PS — IL T WCYCTMTCACT VK1-5 9 3 CASCASTMCVRTRSTT 265 BQ HQ SY DGHNRS AGST FY PS — WR T WCYCTWGGACT VK1-5 9 4 CASCASTMCVRTRSTT 266 BQ HQ SY DGHNRS AGST FY PS PS — T WCYCTYCTACT VK1-5 10 1 CASCASTMCVRTRSTT 267 BQ HQ SY DGHNRS AGST FY PS PLR FY T WCYCTCBTTWCACT VK1-5 10 2 CASCASTMCVRTRSTT 268 BQ HQ SY DGHNRS AGST FY PS PLR IL T WCYCTCBTMTCACT VK1-5 10 3 CASCASTMCVRTRSTT 269 BQ HQ SY DGHNRS AGST FY PS PLR WR T WCYCTCBTWGGACT VK1-12 8 1 CASCASDCTRVCARTT 270 BQ HQ AST ADGNST MS FL — — FY T TSTWCACT VK1-12 8 2 CASCASDCTRVCARTT 271 BQ HQ AST ADGNST MS FL — — IL T TSMTCACT VK1-12 8 3 CASCASDCTRVCARTT 272 BQ HQ AST ADGNST MS FL — — WR T TSWGGACT VK1-12 8 4 CASCASDCTRVCARTT 273 BQ HQ AST ADGNST MS FL P — — T TSCCTACT VK1-12 9 1 CASCASDCTRVCARTT 274 BQ HQ AST ADGNST MS FL P — FY T TSCCTTWCACT VK1-12 9 2 CASCASDCTRVCARTT 275 BQ HQ AST ADGNST MS FL P — IL T TSCCTMTCACT VK1-12 9 3 CASCASDCTRVCARTT 276 BQ HQ AST ADGNST MS FL P — WR T TSCCTWGGACT VK1-12 9 4 CASCASDCTRVCARTT 277 BQ HQ AST ADGNST MS FL P PLR — T TSCCTCBTACT VK1-12 10 1 CASCASDCTRVCARTT 278 BQ HQ AST ADGNST MS FL P PLR FY T TSCCTCBTTWCACT VK1-12 10 2 CASCASDCTRVCARTT 279 BQ HQ AST ADGNST MS FL P PLR IL T TSCCTCBTMTCACT VK1-12 10 3 CASCASDCTRVCARTT 280 BQ HQ AST ADGNST MS FL P PLR WR T TSCCTCBTWGGACT VK1-27 8 1 CASMAGTWCRRTASKG 281 BQ KQ FY DGNS RST AGV — — FY T BATWCACT VK1-27 8 2 CASMAGTWCRRTASKG 282 BQ KQ FY DGNS RST AGV — — IL T BAMTCACT VK1-27 8 3 CASMAGTWCRRTASKG 283 BQ KQ FY DGNS RST AGV — — WR T BAWGGACT VK1-27 8 4 CASMAGTWCRRTASKG 284 BQ KQ FY DGNS RST AGV P — — T BACCTACT VK1-27 9 1 CASMAGTWCRRTASKG 285 BQ KQ FY DGNS RST AGV P — FY T BACCTTWCACT VK1-27 9 2 CASMAGTWCRRTASKG 286 BQ KQ FY DGNS RST AGV P — IL T BACCTMTCACT VK1-27 9 3 CASMAGTWCRRTASKG 287 BQ KQ FY DGNS RST AGV P — WR T BACCTWGGACT VK1-27 9 4 CASMAGTWCRRTASKG 288 BQ KQ FY DGNS RST AGV P PLR — T BACCTCBTACT VK1-27 10 1 CASMAGTWCRRTASKG 289 BQ KQ FY DGNS RST AGV P PLR FY T BACCTCBTTWCACT VK1-27 10 2 CASMAGTWCRRTASKG 290 BQ KQ FY DGNS RST AGV P PLR IL T BACCTCBTMTCACT VK1-27 10 3 CASMAGTWCRRTASKG 291 BQ KQ FY DGNS RST AGV P PLR WR T BACCTCBTWGGACT VK1-33 8 1 CASCWTTMCRATRVCB 292 BQ HL SY DN ADGNST DFH — — FY T WTTWCACT LVY VK1-33 8 2 CASCWTTMCRATRVCB 293 BQ HL SY DN ADGNST DFH — — IL T WTMTCACT LVY VK1-33 8 3 CASCWTTMCRATRVCB 294 BQ HL SY DN ADGNST DFH — — WR T WTWGGACT LVY VK1-33 8 4 CASCWTTMCRATRVCB 295 BQ HL SY DN ADGNST DFH P — - T WTCCTACT LVY VK1-33 9 1 CASCWTTMCRATRVCB 296 BQ HL SY DN ADGNST DFH P — FY T WTCCTTWCACT LVY VK1-33 9 2 CASCWTTMCRATRVCB 297 BQ HL SY DN ADGNST DFH P — IL T WTCCTMTCACT LVY VK1-33 9 3 CASCWTTMCRATRVCB 298 BQ HL SY DN ADGNST DFH P — WR T WTCCTWGGACT LVY VK1-33 9 4 CASCWTTMCRATRVCB 299 BQ HL SY DN ADGNST DFH P PLR — T WTCCTCBTACT LVY VK1-33 10 1 CASCWTTMCRATRVCB 300 BQ HL SY DN ADGNST DFH P PLR FY T WTCCTCBTTWCACT LVY VK1-33 10 2 CASCWTTMCRATRVCB 301 BQ HL SY DN ADGNST DFH P PLR IL T WTCCTCBTMTCACT LVY VK1-33 10 3 CASCWTTMCRATRVCB 302 BQ HL SY DN ADGNST DFH P PLR WR T WTCCTCBTWGGACT LVY VK1-39 8 1 CWGSAAWCATHCMVTA 303 LQ EQ ST FSY HNPRST IST — — FY T BTTWCACT VK1-39 8 2 CWGSAAWCATHCMVTA 304 LQ EQ ST FSY HNPRST IST — — IL T BTMTCACT VK1-39 8 3 CWGSAAWCATHCMVTA 305 LQ EQ ST FSY HNPRST IST — — WR T BTWGGACT VK1-39 8 4 CWGSAAWCATHCMVTA 306 LQ EQ ST FSY HNPRST IST P — — T BTCCTACT VK1-39 9 1 CWGSAAWCATHCMVTA 307 LQ EQ ST FSY HNPRST IST P — FY T BTCCTTWCACT VK1-39 9 2 CWGSAAWCATHCMVTA 308 LQ EQ ST FSY HNPRST IST P — IL T BTCCTMTCACT VK1-39 9 3 CWGSAAWCATHCMVTA 309 LQ EQ ST FSY HNPRST IST P — WR T BTCCTWGGACT VK1-39 9 4 CWGSAAWCATHCMVTA 310 LQ EQ ST FSY HNPRST IST P PLR — T BTCCTCBTACT VK1-39 10 1 CWGSAAWCATHCMVTA 311 LQ EQ ST FSY HNPRST IST P PLR FY T BTCCTCBTTWCACT VK1-39 10 2 CWGSAAWCATHCMVTA 312 LQ EQ ST FSY HNPRST IST P PLR IL T BTCCTCBTMTCACT VK1-39 10 3 CWGSAAWCATHCMVTA 313 LQ EQ ST FSY HNPRST IST P PLR WR T BTCCTCBTWGGACT VK3-11 8 1 CASCASAGWRGKRVCT 314 BQ HQ RS GRS ADGNST SW — — FY T SGTWCACT VK3-11 8 2 CASCASAGWRGKRVCT 315 BQ HQ RS GRS ADGNST SW — — IL T SGMTCACT VK3-11 8 3 CASCASAGWRGKRVCT 316 BQ HQ RS GRS ADGNST SW — — WR T SGWGGACT VK3-11 8 4 CASCASAGWRGKRVCT 317 BQ HQ RS GRS ADGNST SW P — - T SGCCTACT VK3-11 9 1 CASCASAGWRGKRVCT 318 BQ HQ RS GRS ADGNST SW P — FY T SGCCTTWCACT VK3-11 9 2 CASCASAGWRGKRVCT 319 BQ HQ RS GRS ADGNST SW P — IL T SGCCTMTCACT VK3-11 9 3 CASCASAGWRGKRVCT 320 BQ HQ RS GRS ADGNST SW P — WR T SGCCTWGGACT VK3-11 9 4 CASCASAGWRGKRVCT 321 BQ HQ RS GRS ADGNST SW P PLR — T SGCCTCBTACT VK3-11 10 1 CASCASAGWRGKRVCT 322 BQ HQ RS GRS ADGNST SW P PLR FY T SGCCTCBTTWCACT VK3-11 10 2 CASCASAGWRGKRVCT 323 BQ HQ RS GRS ADGNST SW P PLR IL T SGCCTCBTMTCACT VK3-11 10 3 CASCASAGWRGKRVCT 324 BQ HQ RS GRS ADGNST SW P PLR WR T SGCCTCBTWGGACT VK3-15 8 1 CASCASTMCVRTRRKT 325 BQ HQ SY DGHNRS DEGKNRS W — — FY T GGTWCACT VK3-15 8 2 CASCASTMCVRTRRKT 326 BQ HQ SY DGHNRS DEGKNRS W — — IL T GGMTCACT VK3-15 8 3 CASCASTMCVRTRRKT 327 BQ HQ SY DGHNRS DEGKNRS W — — WR T GGWGGACT VK3-15 8 4 CASCASTMCVRTRRKT 328 BQ HQ SY DGHNRS DEGKNRS W P — — T GGCCTACT VK3-15 9 1 CASCASTMCVRTRRKT 329 BQ HQ SY DGHNRS DEGKNRS W P — FY T GGCCTTWCACT VK3-15 9 2 CASCASTMCVRTRRKT 330 BQ HQ SY DGHNRS DEGKNRS W P — IL T GGCCTMTCACT VK3-15 9 3 CASCASTMCVRTRRKT 331 BQ HQ SY DGHNRS DEGKNRS W P — WR T GGCCTWGGACT VK3-15 9 4 CASCASTMCVRTRRKT 332 BQ HQ SY DGHNRS DEGKNRS W P PLR — T GGCCTCBTACT VK3-15 10 1 CASCASTMCVRTRRKT 333 BQ HQ SY DGHNRS DEGKNRS W P PLR FY T GGCCTCBTTWCACT VK3-15 10 2 CASCASTMCVRTRRKT 334 BQ HQ SY DGHNRS DEGKNRS W P PLR IL T GGCCTCBTMTCACT VK3-15 10 3 CASCASTMCVRTRRKT 335 BQ HQ SY DGHNRS DEGKNRS W P PLR WR T GGCCTCBTWGGACT VK3-20 8 1 CASCASTWCGRTRVKK 336 BQ HQ FY DG ADEGK AS — — FY T CATWCACT NRST VK3-20 8 2 CASCASTWCGRTRVKK 337 BQ HQ FY DG ADEGK AS — — IL T CAMTCACT NRST VK3-20 8 3 CASCASTWCGRTRVKK 338 BQ HQ FY DG ADEGK AS — — WR T CAWGGACT NRST VK3-20 8 4 CASCASTWCGRTRVKK 339 BQ HQ FY DG ADEGK AS P — — T CACCTACT NRST VK3-20 9 1 CASCASTWCGRTRVKK 340 BQ HQ FY DG ADEGK AS P — FY T CACCTTWCACT NRST VK3-20 9 2 CASCASTWCGRTRVKK 341 BQ HQ FY DG ADEGK AS P — IL T CACCTMTCACT NRST VK3-20 9 3 CASCASTWCGRTRVKK 342 BQ HQ FY DG ADEGK AS P — WR T CACCTWGGACT NRST VK3-20 9 4 CASCASTWCGRTRVKK 343 BQ HQ FY DG ADEGK AS P PLR — T CACCTCBTACT NRST VK3-20 10 1 CASCASTWCGRTRVKK 344 BQ HQ FY DG ADEGK AS P PLR FY T CACCTCBTTWCACT NRST VK3-20 10 2 CASCASTWCGRTRVKK 345 BQ HQ FY DG ADEGK AS P PLR IL T CACCTCBTMTCACT NRST VK3-20 10 3 CASCASTWCGRTRVKK 346 BQ HQ FY DG ADEGK AS P PLR WR T CACCTCBTWGGACT NRST VK2-28 8 1 ATGCASRBTCKTSASA 347 M HQ AGI LR DEHQ IST — — FY T BTTWCACT STV VK2-28 8 2 ATGCASRBTCKTSASA 348 M HQ AGI LR DEHQ IST — — IL T BTMTCACT STV VK2-28 8 3 ATGCASRBTCKTSASA 349 M HQ AGI LR DEHQ IST — — WR T BTWGGACT STV VK2-28 8 4 ATGCASRBTCKTSASA 350 M HQ AGI LR DEHQ IST P — — T BTCCTACT STV VK2-28 9 1 ATGCASRBTCKTSASA 351 M HQ AGI LR DEHQ IST P — FY T BTCCTTWCACT STV VK2-28 9 2 ATGCASRBTCKTSASA 352 M HQ AGI LR DEHQ IST P — IL T BTCCTMTCACT STV VK2-28 9 3 ATGCASRBTCKTSASA 353 M HQ AGI LR DEHQ IST P — WR T BTCCTWGGACT STV VK2-28 9 4 ATGCASRBTCKTSASA 354 M HQ AGI LR DEHQ IST P PLR — T BTCCTCBTACT STV VK2-28 10 1 ATGCASRBTCKTSASA 355 M HQ AGI LR DEHQ IST P PLR FY T BTCCTCBTTWCACT STV VK2-28 10 2 ATGCASRBTCKTSASA 356 M HQ AGI LR DEHQ IST P PLR IL T BTCCTCBTMTCACT STV VK2-28 10 3 ATGCASRBTCKTSASA 357 M HQ AGI LR DEHQ IST P PLR WR T BTCCTCBTWGGACT STV VK4-1 8 1 CASCASTWCTWCRVCA 358 BQ HQ FY FY ADGNST IST — — FY T BTTWCACT VK4-1 8 2 CASCASTWCTWCRVCA 359 BQ HQ FY FY ADGNST IST — — IL T BTMTCACT VK4-1 8 3 CASCASTWCTWCRVCA 360 BQ HQ FY FY ADGNST IST — — WR T BTWGGACT VK4-1 8 4 CASCASTWCTWCRVCA 361 BQ HQ FY FY ADGNST IST P — — T BTCCTACT VK4-1 9 1 CASCASTWCTWCRVCA 362 BQ HQ FY FY ADGNST IST P — FY T BTCCTTWCACT VK4-1 9 2 CASCASTWCTWCRVCA 363 BQ HQ FY FY ADGNST IST P — IL T BTCCTMTCACT VK4-1 9 3 CASCASTWCTWCRVCA 364 BQ HQ FY FY ADGNST IST P — WR T BTCCTWGGACT VK4-1 9 4 CASCASTWCTWCRVCA 365 BQ HQ FY FY ADGNST IST P PLR — T BTCCTCBTACT VK4-1 10 1 CASCASTWCTWCRVCA 366 BQ HQ FY FY ADGNST IST P PLR FY T BTCCTCBTTWCACT VK4-1 10 2 CASCASTWCTWCRVCA 367 BQ HQ FY FY ADGNST IST P PLR IL T BTCCTCBTMTCACT VK4-1 10 3 CASCASTWCTWCRVCA 368 BQ HQ FY FY ADGNST IST P PLR WR T BTCCTCBTWGGACT [Alternative for VK1-33] (2) VK1-33 8 1 CASCWATMCRATRVCB 369 BQ QL SY DN ADGNST DFH — — FY T WTTWCACT LVY VK1-33 8 2 CASCWATMCRATRVCB 370 BQ QL SY DN ADGNST DFH — — IL T WTMTCACT LVY VK1-33 8 3 CASCWATMCRATRVCB 371 BQ QL SY DN ADGNST DFH — — WR T WTWGGACT LVY VK1-33 8 4 CASCWATMCRATRVCB 372 BQ QL SY DN ADGNST DFH P — — T WTCCTACT LVY VK1-33 9 1 CASCWATMCRATRVCB 373 BQ QL SY DN ADGNST DFH P — FY T WTCCTTWCACT LVY VK1-33 9 2 CASCWATMCRATRVCB 374 BQ QL SY DN ADGNST DFH P — IL T WTCCTMTCACT LVY VK1-33 9 3 CASCWATMCRATRVCB 375 BQ QL SY DN ADGNST DFH P — WR T WTCCTWGGACT LVY VK1-33 9 4 CASCWATMCRATRVCB 376 BQ QL SY DN ADGNST DFH P PLR — T WTCCTCBTACT LVY VK1-33 10 1 CASCWATMCRATRVCB 377 BQ QL SY DN ADGNST DFH P PLR FY T WTCCTCBTTWCACT LVY VK1-33 10 2 CASCWATMCRATRVCB 378 BQ QL SY DN ADGNST DFH P PLR IL T WTCCTCBTMTCACT LVY VK1-33 10 3 CASCWATMCRATRVCB 379 BQ QL SY DN ADGNST DFH P PLR WR T WTCCTCBTWGGACT LVY (1) Junction type 1 has position 96 as FY, type 2 as IL, type 3 as RW, and type 4 has a deletion. (2) Two embodiments are shown for the VK1-33 library. In one embodiment, the second codon was CWT. In another embodiment, it was CWA or CWG.

Example 6.3 More Complex VKCDR3 Libraries

This example demonstrates how a more faithful representation of amino acid variation at each position may be obtained by using a codon-based synthesis approach (Virnekas et al. Nucleic Acids Res., 1994, 22: 5600). This synthetic scheme also allows for finer control of the proportions of particular amino acids included at a position. For example, as described above for the VK1-39 sequences, position 89 was designed as 50% Q and 50% L; however, as Table 30 shows, Q is used much more frequently than L. The more complex VKCDR3 libraries of the present example account for the different relative occurrence of Q and L, for example, 90% Q and 10% L. Such control is better exercised within codon-based synthetic schemes, especially when multiple amino acid types are considered.

This example also describes an implementation of a codon-based synthetic scheme, using the ten VK chassis described in Table 11. Similar approaches, of course, can be implemented with more or fewer such chassis. As indicated in the Detailed Description, a unique aspect of the design of the present libraries, as well as those of the preceding examples, is the germline or chassis-based aspect, which is meant to preserve more of the integrity and variation of actual human kappa light chain sequences. This is in contrast to other codon-based synthesis or degenerate oligonucleotide synthesis approaches that have been described in the literature and that aim to produce “one-size-fits-all” (e.g., consensus) kappa light chain libraries (e.g., Knappik, et al., J Mol Biol, 2000, 296: 57; Akamatsu et al, J Immunol, 1993, 151: 4651).

With reference to Table 30, obtained for VK1-39, one can thus design the length nine VKCDR3 library of Table 34. Here, for practical reasons, the proportions at each position are denoted in multiples of five percentage points. As better synthetic schemes are developed, finer resolution may be obtained—for example to resolutions of one, two, three, or four percent.

TABLE 34 Amino Acid Composition (%) at Each VKCDR3 Position for VK1-39 Library With CDR Length of Nine Residues Amino Acid 89 90 91 92 93 94 95 96 (*) 97 (*) A 5 5 D 5 5 E 5 5 F 5 10 G 5 5 5 5 H 5 5 5 5 I 5 5 K 5 L 10 5 10 20 M N 0 0 5 0 5 P 5 85 5 Q 85 90 5 R 5 5 10 S 80  5 60 5 5 T 10  10 65  90 V 5 W 15 Y 5 75  5 15 Number 3 3 4 6 8 8 3 11 3 Different (*) The composition of positions 96 and 97, determined largely by junction and IGKJ diversity, could be the same for length 9 VK CDR3 of all chassis.

The library of Table 34 would have 1.37×10⁶ unique polypeptide sequences, calculated by multiplying together the numbers in the bottom row of the table.

The underlined 0 entries for Asn (N) at certain positions represent regions where the possibility of having N-linked glycosylation sites in the VKCDR3 has been minimized or eliminated. Peptide sequences with the pattern N-X-(S or T)-Z, where X and Z are different from P, may undergo post-translational modification in a number of expression systems, including yeast and mammalian cells. Moreover, the nature of such modification depends on the specific cell type and, even for a given cell type, on culture conditions. N-linked glycosylation may be disadvantageous when it occurs in a region of the antibody molecule likely to be involved in antigen binding (e.g., a CDR), as the function of the antibody may then be influenced by factors that may be difficult to control. For example, considering position 91 above, one can observe that position 92 is never P. Position 94 is not P in 95% of the cases. However, position 93 is S or T in 75% (65+10) of the cases. Thus, allowing N at position 91 would generate the undesirable motif N-X-(T/S)-Z (with both X and Z distinct from P), and a zero occurrence has therefore been implemented, even though N is observed with some frequency in actual human sequences (see Table 30). A similar argument applies for N at positions 92 and 94. It should be appreciated, however, that if the antibody library were to be expressed in a system incapable of N-linked glycosylation, such as bacteria, or under culture conditions in which N-linked glycosylation did not occur, this consideration may not apply. However, even in the event that the organism used to express libraries with potential N-linked glycosylation sites is incapable of N-linked glycosylation (e.g., bacteria), it may still be desirable to avoid N-X-(S/T) sequences, as the antibodies isolated from such libraries may be expressed in different systems (e.g., yeast, mammalian cells) later (e.g., toward clinical development), and the presence of carbohydrate moieties in the variable domains, and the CDRs in particular, may lead to unwanted modifications of activity. These embodiments are also included within the scope of the invention. To our knowledge, VKCDR3 libraries known in the art have not considered this effect, and thus a proportion of their members may have the undesirable qualities mentioned above.

We also designed additional sub-libraries, related to the library outlined in Table 34, for VKCDR3 of lengths 8 and 10. In these embodiments, the compositions at positions 89 to 94 and 97 remain the same as those depicted in Table 34. Additional diversity, introduced at positions 95 and 95A, the latter being defined for VKCDR3 of length 10 only, are illustrated in Table 35.

TABLE 35 Amino Acid Composition (%) for VK1-39 Libraries of Lengths 8 and 10 Position Position Position 95 - Length 95 - Length 95A - Length Amino Acid 8 (*) 10 (**) 10 A D E F 5 G 5 H I 10 5 K L 20 10 10 M N P 25 85 60 Q R 10 5 10 S 5 5 T 5 V 5 W 10 Y 10 Number 9 3 8 Different (*) Position 96 is deleted in VKCDR3 of size 8. (**) This is the same composition as in VKCDR3 of size 9.

The total number of unique members in the VK1-39 library of length 8, thus, can be obtained as before, and is 3.73×10⁵ (or, 3×3×4×6×8×8×9×3). Similarly, the complexity of the VK1-39 library of length 10 would be 10.9×10⁶ (or 8 times that of the library of size 9, as there is additional 8-fold variation at the insertion position 95A). Thus, there would be a total of 12.7×10⁶ unique members in the overall VK1-39 library, as obtained by summing the number of unique members for each of the specified lengths. In certain embodiments of the invention, it may be preferable to create the individual sub-libraries of lengths 8, 9 and 10 separately, and then mix the sub-libraries in proportions that reflect the length distribution of VKCDR3 in human sequences; for example, in ratios approximating the 1:9:2 distribution that occurs in natural VKCDR3 sequences (see FIG. 3). The present invention provides the compositions and methods for one of ordinary skill synthesizing VKCDR3 libraries corresponding to other VK chassis.

Example 7 A Minimalist VλCDR3 Library

This example describes the design of a minimalist VλCDR3 library. The principles used in designing this library (or more complex Vλ libraries) are similar to those used to design the VKCDR3 libraries. However, unlike the VK genes, the contribution of the IgλV segment to CDRL3 is not constrained to a fixed number of amino acids. Therefore, length variation may be obtained in a minimalist VλCDR3 library even when only considering combinations between Vλ chassis and Jλ sequences.

Examination of the VλCDR3 lengths of human sequences shows that lengths of 9 to 12 account for almost about 95% of sequences, and lengths of 8 to 12 account for about 97% of sequences (FIG. 4). Table 36 shows the usage (percent occurrence) of the six known IGλJ genes in the rearranged human lambda light chain sequences compiled from the NCBI database (see Appendix B), and Table 37 shows the sequences encoded by the genes.

TABLE 36 IGλJ Gene Usage in the Lambda Light Chain Sequences Compiled from the NCBI Database (see Appendix B) Gene_Allele LUA Jλ1_01 20.2% Jλ2_01 42.2% Jλ3_02 36.2% Jλ6_01 0.6% Jλ7_01 0.9%

TABLE 37 Observed Human IGλJ Amino Acid Sequences Gene Sequence SEQ ID NO: IGλJ1-01 YVFGTGTKVTVL 557 IGλJ2-01 VVFGGGTKLTVL 558 IGλJ3-01 WVFGGGTKLTVL 559 IGλJ3-02 VVFGGGTKLTVL 560 IGλJ6-01 NVFGSGTKVTVL 561 IGλJ7-01 AVFGGGTQLTVL 562 IGλJ7-02 AVFGGGTQLTAL 563

IGλJ3-01 and IGλJ7-02 are not represented among the sequences that were analyzed; therefore, they were not included in Table 36. As illustrated in Table 36, IGλJ1-01, IGλJ2-01, and IGλJ3-02 are over-represented in their usage, and have thus been bolded in Table 37. In some embodiments of the invention, for example, only these three over-represented sequences may be utilized. In other embodiments of the invention, one may use all six segments, any 1, 2, 3, 4, or 5 of the 6 segments, or any combination thereof may be utilized.

As shown in Table 14, the portion of CDRL3 contributed by the IGλV gene segment is 7, 8, or 9 amino acids. The remainder of CDRL3 and FRM4 are derived from the IGλJ sequences (Table 37). The IGλJ sequences contribute either one or two amino acids to CDRL3. If two amino acids are contributed by IGλJ, the contribution is from the N-terminal two residues of the IGλJ segment: YV (IGλJ1-01), VV (IGλJ2-01), WV (IGλJ3-01), VV (IGλJ3-02), or AV (IGλJ7-01 and IGλJ7-02). If one amino acid is contributed from IGλJ, it is a V residue, which is formed after the deletion of the N-terminal residue of a IGλJ segment.

In this non-limiting exemplary embodiment of the invention, the FRM4 segment was fixed as FGGGTKLTVL, corresponding to IGλJ2-01 and IGλJ3-02 (i.e., portions of SEQ ID NOs: 558 and 560).

Seven of the 11 selected chassis (Vλ1-40, Vλ3-19, Vλ3-21, Vλ6-57, Vλ1-44, Vλ1-51, and Vλ4-69) have an additional two nucleotides following the last full codon. In four of those seven cases, analysis of the data set provided in Appendix B showed that the addition of a single nucleotide (i.e. without being limited by theory, via the activity of TdT) lead to a further increase in CDRL3 length. This effect can be considered by introducing variants for the L3-Vλ sequences contributed by these four IGλV sequences (Table 38).

TABLE 38 Variants with an additional residue in CDRL3 SEQ CDR3/ ID Name Locus FRM1 CDR1 FRM2 CDR2 FRM3 L3-Vλ NO: 1E+ IGVλ1- QSVLTQPPSVSGAPG TGSSSNIGAG WYQQLPGTAP YGN---- GVPDRFSGSKSG-- QSYDSSL 564 40+ QRVTISC YD---VH KLLI SNRPS TSASLAITGLQAEDEADYYC SG S 3L+ IGVλ3- SSELTQDPAVSVALG QGDSLRSYY- WYQQKPGQAP YGK---- GIPDRFSGSSSG-- NSRDSSG 565 19+ QTVRITC -----AS VLVI NNRPS NTASLTITGAQAEDEADYYC NH H/Q 3H+ IGVλ3- SYVLTQPPSVSVAPG GGNNTGSKS- WYQQKPGQAP YYD---- GIPERFSGSNSG-- QVWDSSS 566 21+ KTARITC -----VH VLVI SDRPS NTATLTISRVEAGDEADYYC DH P 6A+ IGVλ6- NFMLTQPHSVSESPG TRSSGSIASN WYQQRPGSSP YED---- GVPDRFSGSIDSSSNSASLT QSYDSSN 567 57+ KTVTISC Y----VQ TTVI NQRPS ISGLKTEDEADYYC H/Q -- (+) sequences are derived from their parents by the addition of an amino acid at the end of the respective CDR3 (bold underlined). H/Q can be introduced in a single sequence by use of the degenerate codon CAW or similar. Thus, the final set of chassis in the currently exemplified embodiment of the invention is 15: eleven contributed by the chassis in Table 14 and an additional four contributed by the chassis of Table 38. The corresponding L3-VA domains of the 15 chassis contribute from 7 to 10 amino acids to CDRL3. When considering the amino acids contributed by the IGλJ sequences, the total variation in the length of CDRL3 is 8 to 12 amino acids, approximating the distribution in FIG. 4. Thus, in this exemplary embodiment of the invention, the minimalist VP library may be represented by the following: 15 Chassis×5 IGλJ-derived segments=75 sequences. Here, the 15 chassis are Vλ1-40, Vλ1-44, Vλ1-51, Vλ2-14, Vλ3-1*, Vλ3-19, Vλ3-21, Vλ4-69, Vλ6-57, Vλ5-45, Vλ7-43, Vλ1-40+, Vλ3-19+, Vλ3-21+, and Vλ6-57+. The 5 IGλJ-derived segments are YVFGGGTKLTVL (IGλJ1; SEQ ID NO: 568), VVFGGGTKLTVL (IGλJ2; SEQ ID NO: 558), WVFGGGTKLTVL (IGλJ3; SEQ ID NO: 559), AVFGGGTKLTVL (IGλJ7; SEQ ID NO: 569), and -VFGGGTKLTVL (from any of the preceding sequences).

Example 8 Matching to “Reference” Antibodies

CDRH3 sequences of human antibodies of interest that are known in the art, (e.g., antibodies that have been used in the clinic) have close counterparts in the designed library of the invention. A set of fifteen CDRH3 sequences from clinically relevant antibodies is presented in Table 39.

TABLE 39 CDRH3 Sequences of Reference Antibodies SEQ Antibody ID Name Target Origin Status CDHR3 sequence NO: CAB1 TNF-α Phage FDA AKVSYLSTASSLDY 380 display- Approved human library CAB2 EGFR Transgenic FDA VRDRVTGAFDI 381 mouse Approved CAB3 IL-12/IL-23 Phage Phase III KTHGSHDN 382 display- human library CAB4 Interleukin- Transgenic Phase III ARDLRTGPFDY 383 1-β mouse CAB5 RANKL Transgenic Phase III AKDPGTTVIMSWFDP 384 mouse CAB6 IL-12/IL-23 Transgenic Phase III ARRRPGQGYFDF 385 mouse CAB7 TNF-α Transgenic Phase III ARDRGASAGGNYYYYGMDV 386 mouse CAB8 CTLA4 Transgenic Phase III ARDPRGATLYYYYYGMDV 387 mouse CAB9 CD20 Transgenic Phase III AKDIQYGNYYYGMDV 388 mouse CAB10 CD4 Transgenic Phase III ARVINWFDP 389 mouse CAB11 CTLA4 Transgenic Phase III ARTGWLGPFDY 390 mouse CAB12 IGF1-R Transgenic Phase II AKDLGWSDSYYYYYGMDV 391 mouse CAB13 EGFR Transgenic Phase II ARDGITMVRGVMKDYFDY 392 mouse CAB14 EGFR Phage Phase II ARVSIFGVGTFDY 393 display- human library CAB15 BLyS Phage Phase II ARSRDLLLFPHHALSP 394 display- human library

Each of the above sequences was compared to each of the members of the library of Example 5, and the member, or members, with the same length and fewest number of amino acid mismatches was, or were, recorded. The results are summarized in Table 40, below. For most of the cases, matches with 80% identity or better were found in the exemplified CDRH3 library. To the extent that the specificity and binding affinity of each of these antibodies is influenced by their CDRH3 sequence, without being bound by theory, one or more of these library members could have measurable affinity to the relevant targets.

TABLE 40 Match of Reference Antibody CDRH3 to Designed Library Number of % Identity Antibody Name Mismatches (*) Length of Best Match CAB1 5 14 64% CAB2 2 11 82% CAB3 4 8 50% CAB4 2 11 82% CAB5 3 15 80% CAB6 3 12 75% CAB7 2 20 90% CAB8 0 19 100%  CAB9 3 15 80% CAB10 1 9 89% CAB11 1 11 91% CAB12 2 18 89% CAB13 2 18 89% CAB14 1 13 92% CAB15 7 16 56% (*) For the best-matching sequence(s) in library

Given that a physical realization of a library with about 10⁸ distinct members could, in practice, contain every single member, then such sequences with close percent identity to antibodies of interest would be present in the physical realization of the library. This example also highlights one of many distinctions of the libraries of the current invention over those of the art; namely, that the members of the libraries of the invention may be precisely enumerated. In contrast, CDRH3 libraries known in the art cannot be explicitly enumerated in the manner described herein. For example, many libraries known in the art (e.g., Hoet et al., Nat. Biotechnol., 2005, 23: 344; Griffiths et al., EMBO J., 1994, 13: 3245; Griffiths et al., EMBO J., 1993, 12: 725; Marks et al., J. Mol. Biol., 1991, 222: 581, each incorporated by reference in its entirety) are derived by cloning of natural human CDRH3 sequences and their exact composition is not characterized, which precludes enumeration.

Synthetic libraries produced by other (e.g., random or semi-random/biased) methods (Knappik, et al., J Mol Biol, 2000, 296: 57, incorporated by reference in its entirety) tend to have very large numbers of unique members. Thus, while matches to a given input sequence (for example, at 80% or greater) may exist in a theoretical representation of such libraries, the probability of synthesizing and then producing a physical realization of the theoretical library that contains such a sequence and then selecting an antibody corresponding to such a match, in practice, may be remotely small. For example, a CDRH3 of length 19 in the Knappik library may have over 10¹⁹ distinct sequences. In a practical realization of such a library a tenth or so of the sequences may have length 19 and the largest total library may have in the order of 10¹⁰ to 10¹² transformants; thus, the probability of a given pre-defined member being present, in practice, is effectively zero (less than one in ten million). Other libraries (e.g., Enzelberger et al. WO2008053275 and Ladner US20060257937, each incorporated by reference in its entirety) suffer from at least one of the limitations described throughout this application.

Thus, for example, considering antibody CAB14, there are seven members of the designed library of Example 5 that differ at just one amino acid position from the sequence of the CDRH3 of CAB14 (given in Table 39). Since the total length of this CDRH3 sequence is 13, the percent of identical amino acids is 12/13 or about 92% for each of these 7 sequences of the library of the invention. It can be estimated that the probability of obtaining such a match (or better) in the library of Knappik et al. is about 1.4×10⁻⁹; it would be lower still, about 5.5×10⁻¹⁰, in a library with equal amino acid proportions (i.e., completely random). Therefore, in a physical realization of the library with about 10¹⁰ transformants of which about a tenth may have length 13, there may be one or two instances of these best matches. However, with longer sequences such as CAB12, the probability of having members in the Knappik library with about 89% or better matching are under about 10⁻¹⁵, so that the expected number of instances in a physical realization of the library is essentially zero. To the extent that sequences of interest resemble actual human CDRH3 sequences, there will be close matches in the library of Example 5, which was designed to mimic human sequences. Thus, one of the many relative advantages of the present library, versus those in the art, becomes more apparent as the length of the CDRH3 increases.

Example 9 Split Pool Synthesis of Oligonucleotides Encoding the DH, N2, and H3-JH Segments

This example outlines the procedures used to synthesize the oligonucleotides used to construct the exemplary libraries of the invention. Custom Primer Support™ 200 dT40S resin (GE Healthcare) was used to synthesize the oligonucleotides, using a loading of about 39 μmol/g of resin. Columns (diameter=30 μm) and frits were purchased from Biosearch Technologies, Inc. A column bed volume of 30 μL was used in the synthesis, with 120 nmol of resin loaded in each column. A mixture of dichloromethane (DCM) and methanol (MeOH), at a ratio of 400/122 (v/v) was used to load the resin. Oligonucleotides were synthesized using a Dr. Oligo® 192 oligonucleotide synthesizer and standard phosphorothioate chemistry.

The split pool procedure for the synthesis of the [DH]-[N2]-[H3-JH] oligonucleotides was performed as follows: First, oligonucleotide leader sequences, containing a randomly chosen 10 nucleotide sequence (ATGCACAGTT; SEQ ID NO: 395), a BsrDI recognition site (GCAATG), and a two base “overlap sequence” (TG, AC, AG, CT, or GA) were synthesized. The purpose of each of these segments is explained below. After synthesis of this 18 nucleotide sequence, the DH segments were synthesized; approximately 1 g of resin (with the 18 nucleotide segment still conjugated) was suspended in 20 mL of DCM/MeOH. About 60 μL of the resulting slurry (120 nmol) was distributed inside each of 278 oligonucleotide synthesis columns. These 278 columns were used to synthesize the 278 DH segments of Table 18, 3′ to the 18 nucleotide segment described above. After synthesis, the 278 DH segments were pooled as follows: the resin and frits were pushed out of the columns and collected inside a 20 mL syringe barrel (without plunger). Each column was then washed with 0.5 mL MeOH, to remove any residual resin that was adsorbed to the walls of the column. The resin in the syringe barrel was washed three times with MeOH, using a low porosity glass filter to retain the resin. The resin was then dried and weighed.

The pooled resin (about 1.36 g) containing the 278 DH segments was subsequently suspended in about 17 mL of DCM/MeOH, and about 60 μL of the resulting slurry was distributed inside each of two sets of 141 columns. The 141 N2 segments enumerated in Tables 24 and 25 were then synthesized, in duplicate (282 total columns), 3′ to the 278 DH segments synthesized in the first step. The resin from the 282 columns was then pooled, washed, and dried, as described above.

The pooled resin obtained from the N2 synthesis (about 1.35 g) was suspended in about 17 mL of DCM/MeOH, and about 60 μL of the resulting slurry was distributed inside each of 280 columns, representing 28 H3-JH segments synthesized ten times each. A portion (described more fully below) of each of the 28 IGHJ segments, including H3-JH of Table 20 were then synthesized, 3′ to the N2 segments, in ten of the columns. Final oligonucleotides were cleaved and deprotected by exposure to gaseous ammonia (85° C., 2 h, 60 psi).

Split pool synthesis was used to synthesize the exemplary CDRH3 library. However, it is appreciated that recent advances in oligonucleotide synthesis, which enable the synthesis of longer oligonucleotides at higher fidelity and the production of the oligonucleotides of the library by synthetic procedures that involve splitting, but not pooling, may be used in alternative embodiments of the invention. The split pool synthesis described herein is, therefore, one possible means of obtaining the oligonucleotides of the library, but is not limiting. One other possible means of synthesizing the oligonucleotides described in this application is the use of trinucleotides. This may be expected to increase the fidelity of the synthesis, since frame shift mutants would be reduced or eliminated.

Example 10 Construction of the CDRH3 and Heavy Chain Libraries

This example outlines the procedures used to create exemplary CDRH3 and heavy chain libraries of the invention. A two step process was used to create the CDRH3 library. The first step involved the assembly of a set of vectors encoding the tail and N1 segments, and the second step involved utilizing the split pool nucleic acid synthesis procedures outlined in Example 9 to create oligonucleotides encoding the DH, N2, and H3-JH segments. The chemically synthesized oligonucleotides were then ligated into the vectors, to yield CDRH3 residues 95-102, based on the numbering system described herein. This CDRH3 library was subsequently amplified by PCR and recombined into a plurality of vectors containing the heavy chain chassis variants described in Examples 1 and 2. CDRH1 and CDRH2 variants were produced by QuikChange® Mutagenesis (Stratagene™), using the oligonucleotides encoding the ten heavy chain chassis of Example 1 as a template. In addition to the heavy chain chassis, the plurality of vectors contained the heavy chain constant regions (i.e., CH1, CH2, and CH3) from IgG1, so that a full-length heavy chain was formed upon recombination of the CDRH3 with the vector containing the heavy chain chassis and constant regions. In this exemplary embodiment, the recombination to produce the full-length heavy chains and the expression of the full-length heavy chains were both performed in S. cerevisiae.

To generate full-length, heterodimeric IgGs, comprising a heavy chain and a light chain, a light chain protein was also expressed in the yeast cell. The light chain library used in this embodiment was the kappa light chain library, wherein the VKCDR3s were synthesized using degenerate oligonucleotides (see Example 6.2). Due to the shorter length of the oligonucleotides encoding the light chain library (in comparison to those encoding the heavy chain library), the light chain CDR3 oligonucleotides could be synthesized de novo, using standard procedures for oligonucleotide synthesis, without the need for assembly from sub-components (as in the heavy chain CDR3 synthesis). One or more light chains can be expressed in each yeast cell which expresses a particular heavy chain clone from a library of the invention. One or more light chains have been successfully expressed from both episomal (e.g. plasmid) vectors and from integrated sites in the yeast genome.

Below are provided further details on the assembly of the individual components for the synthesis of a CDRH3 library of the invention, and the subsequent combination of the exemplary CDRH3 library with the vectors containing the chassis and constant regions. In this particular exemplary embodiment of the invention, the steps involved in the process may be generally characterized as (i) synthesis of 424 vectors encoding the tail and N1 regions; (ii) ligation of oligonucleotides encoding the [DH]-[N2]-[H3-JH] segments into these 424 vectors; (iii) PCR amplification of the CDRH3 sequences from the vectors produced in these ligations; and (iv) homologous recombination of these PCR-amplified CDRH3 domains into the yeast expression vectors containing the chassis and constant regions.

Example 10.1 Synthesis of Vectors Encoding the Tail and N1 Regions

This example demonstrates the synthesis of 424 vectors encoding the tail and N1 regions of CDRH3. In this exemplary embodiment of the invention, the tail was restricted to G, D, E, or nothing, and the N1 region was restricted to one of the 59 sequences shown in Table 24. As described throughout the specification, many other embodiments are possible.

In the first step of the process, a single “base vector” (pJM204, a pUC-derived cloning vector) was constructed, which contained (i) a nucleic acid sequence encoding two amino acids that are common to the C-terminal portion of all 28 IGHJ segments (SS), and (ii) a nucleic acid sequence encoding a portion of the CH1 constant region from IgG1. Thus, the base vector contains an insert encoding a sequence that can be depicted as:

[SS]-[CH1˜],

wherein SS is a common portion of the C-terminus of the 28 IGHJ segments and CH1˜ is a portion of the CH1 constant region from IgG1, namely:

(SEQ ID NO: 396) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLG.

Next, 424 different oligonucleotides were cloned into the base vector, upstream (i.e., 5′) from the region encoding the [SS]-[CH1˜]. These 424 oligonucleotides were synthesized by standard methods and each encoded a C-terminal portion of one of the 17 heavy chain chassis enumerated in Table 5, plus one of four exemplary tail segments (G/D/E/-), and one of 59 exemplary N1 segments (Table 24). These 424 oligonucleotides, therefore, encode a plurality of sequences that may be represented by:

[˜FRM3]-[G/D/E/-]-[N1],

wherein ˜FRM3 represents a C-terminal portion of a FRM3 region from one of the 17 heavy chain chassis of Table 5, G/D/E/- represents G, D, E, or nothing, and N1 represents one of the 59 N1 sequences enumerated in Table 24. As described throughout the specification, the invention is not limited to the chassis exemplified in Table 5, their CDRH1 and CDRH2 variants (Table 8), the four exemplary tail options used in this example, or the 59 N1 segments presented in Table 24.

The oligonucleotide sequences represented by the sequences above were synthesized in two groups: one group containing a ˜FRM3 region identical to the corresponding region on 16 of the 17 the heavy chain chassis enumerated in Table 5, and another group containing a ˜FRM3 region that is identical to the corresponding region on VH3-15. In the former group, an oligonucleotide encoding DTAVYYCAR (SEQ ID NO: 397) was used for FRM3. During subsequent PCR amplification, the V residue of VH5-51 was altered to an M, to correspond to the VH5-51 germline sequence. In the latter group (that with a sequence common to VH3-15), a larger oligonucleotide, encoding the sequence AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 398) was used for ˜FRM3. Each of the two oligonucleotides encoding the FRM3 regions were paired with oligonucleotides encoding one of the four tail regions (G/D/E/-) and one of the 59 N1 segments, yielding a total of 236 possible combinations for each ˜FRM3 (i.e., 1×4×59), or a total of 472 possible combinations when both FRM3 sequences are considered. However, 48 of these combinations are redundant and only a single representation of these sequences was used in the currently exemplified CDRH3 library, yielding 424 unique oligonucleotides encoding [˜FRM3]-[G/D/E/-]-[N1] sequences.

After the oligonucleotides encoding the [˜FRM3]-[G/D/E/-]-[N1] and [SS]-[CH1˜] segments were cloned into the vector, as described above, additional sequences were added to the vector to facilitate the subsequent insertion of the oligonucleotides encoding the [DH]-[N2]-[H3-JH] fragments synthesized during the split pool synthesis. These additional sequences comprise a polynucleotide encoding a selectable marker protein, flanked on each side by a recognition site for a type II restriction enzyme, for example:

[Type II RS 1]-[selectable marker protein]-[Type II RS 2].

In this exemplary embodiment, the selectable marker protein is ccdB and the type II restriction enzyme recognition sites are specific for BsrDI and BbsI. In certain strains of E. coli, the ccdB protein is toxic, thereby preventing the growth of these bacteria when the gene is present.

An example of the 5′ end of one of the 212 vectors with a ˜FRM3 region based on the VH3-23 chassis, D tail residue and an N1 segment of length zero is presented below (SEQ ID NO: 570):

                                                                    VH3-23                                                          ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~                                                           A  I  S   G  S  G   G  S  T  Y •  961                                                          GCTATTAG TGGTAGTGGT GGTAGCACAT                                                          CGATAATC ACCATCACCA CCATCGTGTA                                         VH3-23 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ •  Y  A  D   S  V  K   G  R  F  T   I  S  R   D  N  S   K  N  T  L   Y  L  Q   M  N  S 1041 ACTACGCAGA CTCCGTGAAG GGCCGGTTCA CCATCTCCAG AGACAATTCC AAGAACACGC TGTATCTGCA AATGAACAGC TGATGCGTCT GAGGCACTTC CCGGCCAAGT GGTAGAGGTC TCTGTTAAGG TTCTTGTGCG ACATAGACGT TTACTTGTCG                 VH3-23                                            ccdB ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~                ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~                                               BsrDI                                               ~~~~~~  L  R  A  E   D  T  A   V  Y  Y   C  A  K 1121 CTGAGAGCCG AGGACACGGC GGTGTACTAC TGCGCCAAGG ACCATTGCGC TTAGCCTAGG TTATATTCCC CAGAACATCA GACTCTCGGC TCCTGTGCCG CCACATGATG ACGCGGTTCC TGGTAACGCG AATCGGATCC AATATAAGGG GTCTTGTAGT

An example of one of the 212 vectors with a ˜FRM3 region based on one of the other 16 chassis, with a D residue as the tail and an N1 segment of length zero is presented below (SEQ ID NO: 571):

                                                                Framework 3                                                         ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~                                                          D  T  A   V  Y  Y  C   A  R  961                                                         GACACGGCG GTGTACTACT GCGCCAGAGA                                                         CTGTGCCGC CACATGATGA CGCGGTCTCT                                   ccdB  ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  BsrDI  ~~~~~~ 1041 CCATTGCGCT TAGCCTAGGT TATATTCCCC AGAACATCAG GTTAATGGCG TTTTTGATGT CATTTTCGCG GTGGCTGAGA GGTAACGCGA ATCGGATCCA ATATAAGGGG TCTTGTAGTC CAATTACCGC AAAAACTACA GTAAAAGCGC CACCGACTCT                                   ccdB ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1121 TCAGCCACTT CTTCCCCGAT AACGGAAACC GGCACACTGG CCATATCGGT GGTCATCATG CGCCAGCTTT CATCCCCGAT AGTCGGTGAA GAAGGGGCTA TTGCCTTTGG CCGTGTGACC GGTATAGCCA CCAGTAGTAC GCGGTCGAAA GTAGGGGCTA                                   ccdB ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1201 ATGCACCACC GGGTAAAGTT CACGGGAGAC TTTATCTGAC AGCAGACGTG CACTGGCCAG GGGGATCACC ATCCGTCGCC TACGTGGTGG CCCATTTCAA GTGCCCTCTG AAATAGACTG TCGTCTGCAC GTGACCGGTC CCCCTAGTGG TAGGCAGCGG                                   ccdB ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1281 CGGGCGTGTC AATAATATCA CTCTGTACAT CCACAAACAG ACGATAACGG CTCTCTCTTT TATAGGTGTA AACCTTAAAC GCCCGCACAG TTATTATAGT GAGACATGTA GGTGTTTGTC TGCTATTGCC GAGAGAGAAA ATATCCACAT TTGGAATTTG                                   ccdB ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1361 TGCATTTCAC CAGCCCCTGT TCTCGTCAGC AAAAGAGCCG TTCATTTCAA TAAACCGGGC GACCTCAGCC ATCCCTTCCT ACGTAAAGTG GTCGGGGACA AGAGCAGTCG TTTTCTCGGC AAGTAAAGTT ATTTGGCCCG CTGGAGTCGG TAGGGAAGGA                                   ccdB ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1441 GATTTTCCGC TTTCCAGCGT TCGGCACGCA GACGACGGGC TTCATTCTGC ATGGTTGTGC TTACCAGACC GGAGATATTG CTAAAAGGCG AAAGGTCGCA AGCCGTGCGT CTGCTGCCCG AAGTAAGACG TACCAACACG AATGGTCTGG CCTCTATAAC                                   ccdB ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1521 ACATCATATA TGCCTTGAGC AACTGATAGC TGTCGCTGTC AACTGTCACT GTAATACGCT GCTTCATAGC ATACCTCTTT TGTAGTATAT ACGGAACTCG TTGACTATCG ACAGCGACAG TTGACAGTGA CATTATGCGA CGAAGTATCG TATGGAGAAA                                   ccdB ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1601 TTGACATACT TCGGGTATAC ATATCAGTAT ATATTCTTAT ACCGCAAAAA TCAGCGCGCA AATATGCATA CTGTTATCTG AACTGTATGA AGCCCATATG TATAGTCATA TATAAGAATA TGGCGTTTTT AGTCGCGCGT TTATACGTAT GACAATAGAC        ccdB                                       CH1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~        ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~                               BbsI                              ~~~~~~~                                            A   S  T  K   G  P  S   V  F  P  L   A  P  S • 1681 GCTTTTAGTA AGCCGCCTAG GTCATCAGAA GACAACTCAG CTAGCACCAA GGGCCCATCG GTCTTTCCCC TGGCACCCTC CGAAAATCAT TCGGCGGATC CAGTAGTCTT CTGTTGAGTC GATCGTGGTT CCCGGGTAGC CAGAAAGGGG ACCGTGGGAG                                       CH1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ • S  K  S   T  S  G  G   T  A  A   L  G  C   L  V  K  D   Y  F  P   E  P  V   T  V  S  W • 1761 CTCCAAGAGC ACCTCTGGGG GCACAGCGGC CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT GAGGTTCTCG TGGAGACCCC CGTGTCGCCG GGACCCGACG GACCAGTTCC TGATGAAGGG GCTTGGCCAC TGCCACAGCA                                CH1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ •  N  S  G   A  L  T   S  G  V  H   T  F  P   A  V  L   Q  S  S  G   L 1841 GGAACTCAGG CGCCCTGACC AGCGGCGTGC ACACCTTCCC GGCTGTCCTA CAGTCCTCAG GACTC CCTTGAGTCC GCGGGACTGG TCGCCGCACG TGTGGAAGGG CCGACAGGAT GTCAGGAGTC CTGAG

All 424 vectors were sequence verified. A schematic diagram of the content of the 424 vectors, before and after cloning of the [DH]-[N2]-[H3-JH] fragment is presented in FIG. 5. Below is an exemplary sequence from one of the 424 vectors containing a FRM3 region from VH3-23 (SEQ ID NO: 572).

                                                      primer EMK135                                                   ~~~~~~~~~~~~~~~~~~~~~~~~                                                   VH3-23                           ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~                           A  I  S   G  S  G   G  S  T   Y  Y  A  D   S  V  K   G  R  F  561                          GCTATTA GTGGTAGTGG TGGTAGCACA TACTACGCAG ACTCCGTGAA GGGCCGGTTC                          CGATAAT CACCATCACC ACCATCGTGT ATGATGCGTC TGAGGCACTT CCCGGCCAAG                                      VH3-23 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  T  I  S  R   D  N  S   K  N  T   L  Y  L  Q   M  N  S   L  R  A   E  D  T  A   V  Y  Y •  641 ACCATCTCCA GAGACAATTC CAAGAACACG CTGTATCTGC AAATGAACAG CCTGAGAGCC GAGGACACGG CGGTGTACTA TGGTAGAGGT CTCTGTTAAG GTTCTTGTGC GACATAGACG TTTACTTGTC GGACTCTCGG CTCCTGTGCC GCCACATGAT   VH3-23                              D                                    J1 ~~~~~~~~~~~             ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~          ~~~~~~~~~~~~~~~~~~~~                                                                            JH6                                                                    ~~~~~~~~~~~~~~~~~~~~                N1_9                                         N2            ~~~~~~~~~~~~~                                 ~~~~~~~~~~ • C  A  K   D  A  G  G   Y  Y  Y   G  S  G   S  Y  Y  N   A  A  A   Y  Y  Y   Y  Y  G  M •  721 CTGCGCCAAG GACGCCGGAG GATATTATTA TGGGTCAGGA AGCTATTACA ACGCTGCGGC TTACTACTAC TATTATGGCA GACGCGGTTC CTGCGGCCTC CTATAATAAT ACCCAGTCCT TCGATAATGT TGCGACGCCG AATGATGATG ATAATACCGT                    JH6 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~    J1                                                          CH1 ~~~~~~~~                                     ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~                                               NheI                                              ~~~~~~ •  D  V  W   G  Q  G   T  T  V  T   V  S  S   A  S  T   K  G  P  S   V  F  P   L  A  P  801 TGGACGTGTG GGGACAAGGT ACAACAGTCA CCGTCTCCTC AGCTAGCACC AAGGGCCCAT CGGTCTTTCC CCTGGCACCC ACCTGCACAC CCCTGTTCCA TGTTGTCAGT GGCAGAGGAG TCGATCGTGG TTCCCGGGTA GCCAGAAAGG GGACCGTGGG                                       CH1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  S  S  K  S   T  S  G   G  T  A   A  L  G  C   L  V  K   D  Y  F   P  E  P  V   T  V  S •  881 TCCTCCAAGA GCACCTCTGG GGGCACAGCG GCCCTGGGCT GCCTGGTCAA GGACTACTTC CCCGAACCGG TGACGGTGTC AGGAGGTTCT CGTGGAGACC CCCGTGTCGC CGGGACCCGA CGGACCAGTT CCTGATGAAG GGGCTTGGCC ACTGCCACAG                                        EK137 CH1 Primer                                       ~~~~~~~~~~~~~~~~~~~~                                       CH1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ • W  N  S   G  A  L  T   S  G  V   H  T  F   P  A  V  L   Q  S  S   G  L  Y   S  L  S  S •  961 GTGGAACTCA GGCGCCCTGA CCAGCGGCGT GCACACCTTC CCGGCTGTCC TACAGTCCTC AGGACTCTAC TCCCTCAGCA CACCTTGAGT CCGCGGGACT GGTCGCCGCA CGTGTGGAAG GGCCGACAGG ATGTCAGGAG TCCTGAGATG AGGGAGTCGT               CH1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ •  V  V  T   V  P  S   S  S  L  G 1041 GCGTGGTGAC CGTGCCCTCC AGCAGCTTGG GC CGCACCACTG GCACGGGAGG TCGTCGAACC CG

Example 10.2 Cloning of the Oligonucleotides Encoding the DH, N2, H3-JH Segments into the Vectors Containing the Tail and N1 Segments

This example describes the cloning of the oligonucleotides encoding the [D]-[N2]-[H3-JH] segments (made via split pool synthesis; Example 9) into the 424 vectors produced in Example 10.1. To summarize, the [DH]-[N2]-[H3-JH] oligonucleotides produced via split pool synthesis were amplified by PCR, to produce double-stranded oligonucleotides, to introduce restriction sites that would create overhangs complementary to those on the vectors (i.e., BsrDI and BbsI), and to complete the 3′ portion of the IGHJ segments that was not synthesized in the split pool synthesis. The amplified oligonucleotides were then digested with the restriction enzymes BsrDI (cleaves adjacent to the DH segment) and BbsI (cleaves near the end of the JH segment). The cleaved oligonucleotides were then purified and ligated into the 424 vectors which had previously been digested with BsrDI and BbsI. After ligation, the reactions were purified, ethanol precipitated, and resolubilized.

This process for one of the [DH]-[N2]-[H3-JH] oligonucleotides synthesized in the split pool synthesis is illustrated below. The following oligonucleotide (SEQ ID NO: 399) is one of the oligonucleotides synthesized during the split pool synthesis:

 1 ATGCACAGTTGCAATG TG TATTACTATGGATCTGGTTCTTACTATAAT GT 50 51 GGGCGGA TATTATTACTACTATGGTATGGACGTATGGGGGCAAGGGACC 99

The first 10 nucleotides (ATGCACAGTT; SEQ ID NO: 395) represent a portion of a random sequence that is increased to 20 base pairs in the PCR amplification step, below. This portion of the sequence increases the efficiency of BsrDI digestion and facilitates the downstream purification of the oligonucleotides.

Nucleotides 11-16 (underlined) represent the BsrDI recognition site. The two base overlap sequence that follows this site (in this example TG; bold) was synthesized to be complementary to the two base overhang created by digesting certain of the 424 vectors with BsrDI (i.e., depending on the composition of the tail/N1 region of the particular vector). Other oligonucleotides contain different two-base overhangs, as described below.

The two base overlap is followed by the DH gene segment (nucleotides 19-48), in this example, by a 30 bp sequence (TATTACTATGGATCTGGTTCTTACTATAAT, SEQ ID NO: 400) which encodes the ten residue DH segment YYYGSGSYYN (i.e., IGHD3-10_(—)2 of Table 17; SEQ ID NO: 2).

The region of the oligonucleotide encoding the DH segment is followed, in this example, by a nine base region (GTGGGCGGA; bold; nucleotides 49-57), encoding the N2 segment (in this case VGG; Table 24).

The remainder of this exemplary oligonucleotide represents the portion of the JH segment that is synthesized during the split pool synthesis (TATTATTACTACTATGGTATGGACGTATGGGGGCAAGGGACC; SEQ ID NO: 401; nucleotides 58-99; underlined), encoding the sequence YYYYYGMDVWGQGT (Table 20; residues 1-14 of SEQ ID NO: 258). The balance of the IGHJ segment is added during the subsequent PCR amplification described below.

After the split pool-synthesized oligonucleotides were cleaved from the resin and deprotected, they served as a template for a PCR reaction which added an additional randomly chosen 10 nucleotides (e.g., GACGAGCTTC; SEQ ID NO: 402) to the 5′ end and the rest of the IGHJ segment plus the BbsI restriction site to the 3′ end. These additions facilitate the cloning of the [DH]-[N2]-[JH] oligonucleotides into the 424 vectors. As described above (Example 9), the last round of the split pool synthesis involves 280 columns: 10 columns for each of the oligonucleotides encoding one of 28 H3-JH segments. The oligonucleotide products obtained from these 280 columns are pooled according to the identity of their H3-JH segments, for a total of 28 pools. Each of these 28 pools is then amplified in five separate PCR reactions, using five forward primers that each encode a different two base overlap (preceding the DH segment; see above) and one reverse primer that has a sequence corresponding to the familial origin of the H3-JH segment being amplified. The sequences of these 11 primers are provided below:

Forward primers AC GACGAGCTTCAATGCACAGTTGCAATGAC (SEQ ID NO: 403) AG GACGAGCTTCAATGCACAGTTGCAATGAG (SEQ ID NO: 404) CT GACGAGCTTCAATGCACAGTTGCAATGCT (SEQ ID NO: 405) GA GACGAGCTTCAATGCACAGTTGCAATGGA (SEQ ID NO: 406) TG GACGAGCTTCAATGCACAGTTGCAATGTG (SEQ ID NO: 407) Reverse Primers JH1 TGCATCAGTGCGACTAACGGAAGACTCTGAGGAGACGGTGACCAAGGTGCCCTGGCCCCA (SEQ ID NO: 408) JH2 TGCATCAGTGCGACTAACGGAAGACTCTGAGGAGACAGTGACCAAGGTGCCACGGCCCCA (SEQ ID NO: 409) JH3 TGCATCAGTGCGACTAACGGAAGACTCTGAAGAGACGGTGACCATTGTCCCTTGGCCCCA (SEQ ID NO: 410) JH4 TGCATCAGTGCGACTAACGGAAGACTCTGAGGAGACGGTGACCAAGGTTCCTTGGCCCCA (SEQ ID NO: 411) JH5 TGCATCAGTGCGACTAACGGAAGACTCTGAGGAGACGGTGACCAAGGTTCCCTGGCCCCA (SEQ ID NO: 412) JH6 TGCATCAGTGCGACTAACGGAAGACTCTGAGGAGACGGTGACCGTGGTCCCTTGCCCCCA (SEQ ID NO: 413)

Amplifications were performed using Taq polymerase, under standard conditions. The oligonucleotides were amplified for eight cycles, to maintain the representation of sequences of different lengths. Melting of the strands was performed at 95° C. for 30 seconds, with annealing at 58° C. and a 15 second extension time at 72° C.

Using the exemplary split-pool derived oligonucleotide enumerated above as an example, the PCR amplification was performed using the TG primer and the JH6 primer, where the annealing portion of the primers has been underlined:

TG GACGAGCTTCAATGCACAGTTGCAATGTG (SEQ ID NO: 407) JH6 TGCATCAGTGCGACTAACGGAAGACTCTGAGGAGACGGTGACCGTGGTCCCTTGCCCCCA (SEQ ID NO: 413) The portion of the TG primer that is 5′ to the annealing portion includes the random 10 base pairs described above. The portion of the JH6 primer that is 5′ to the annealing portion includes the balance of the JH6 segment and the BbsI restriction site. The following PCR product (SEQ ID NO: 414) is formed in the reaction (added sequences underlined):

GACGAGCTTCATGCACAGTTGCAATGTGTATTACTATGGATCTGGTTCTT ACTATAATGTGGGCGGATATTATTACTACTATGGTATGGACGTATGGGGG CAAGGGACCACGGTCACCGTCTCCTCAGAGTCTTCCGTTAGTCGCACTGA TGCAG

The PCR products from each reaction were then combined into five pools, based on the forward primer that was used in the reaction, creating sets of sequences yielding the same two-base overhang after BsrDI digestion. The five pools of PCR products were then digested with BsRDI and BbsI (100 μg of PCR product; 1 mL reaction volume; 200 U BbsI; 100 U BsrDI; 2 h; 37° C.; NEB Buffer 2). The digested oligonucleotides were extracted twice with phenol/chloroform, ethanol precipitated, air dried briefly and resolubilized in 300 μL of TE buffer by sitting overnight at 4° C.

Each of the 424 vectors described in the preceding sections was then digested with BsrDI and BbsI, each vector yielding a two base overhang that was complimentary to one of those contained in one of the five pools of PCR products. Thus, one of the five pools of restriction digested PCR products are ligated into each of the 424 vectors, depending on their compatible ends, for a total of 424 ligations.

Example 10.3 PCR Amplification of the CDRH3 from the 424 Vectors

This example describes the PCR amplification of the CDRH3 regions from the 424 vectors described above. As set forth above, the 424 vectors represent two sets: one for the VH3-23 family, with FRM3 ending in CAK (212 vectors) and one for the other 16 chassis, with FRM3 ending in CAR (212 vectors). The CDRH3s in the VH3-23-based vectors were amplified using a reverse primer (EK137; see Table 41) recognizing a portion of the CH1 region of the plasmid and the VH3-23-specific primer EK135 (see Table 41). Amplification of the CDRH3s from the 212 vectors with FRM3 ending in CAR was performed using the same reverse primer (EK137) and each of five FRM3-specific primers shown in Table 41 (EK139, EK140, EK141, EK143, and EK144). Therefore, 212 VH3-23 amplifications and 212×5 FRM3 PCR reactions were performed, for a total of 1,272 reactions. An additional PCR reaction amplified the CDRH3 from the 212 VH3-23-based vectors, using the EK 133 forward primer, to allow the amplicons to be cloned into the other 5 VH3 family member chassis while making the last three amino acids of these chassis CAK instead of the original CAR (VH3-23*). The primers used in each reaction are shown in Table 41.

TABLE 41 Primers Used for Amplification of CDRH3 Sequences SEQ Primer Compatible ID No. Chassis Primer Sequence NO EK135 VH3-23 CACATACTACGCAGACTCCGTG 415 EK133 VH3-48; CAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTG 416 VH3-7; VH3-15; VH3-30; VH3-33; VH3-23* EK139 VH4-B; AAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTG 417 VH4-31; VH4-34; VH4-39; VH4-59; VH4-61 EK140 VH1-46; GAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTG 418 VH1-69 EK141 VH1-2 GAGCTGAGCAGGCTGAGATCTGACGACACGGCGGTGTACTACTG 419 EK143 VH5-51 CAGTGGAGCAGCCTGAAGGCCTCGGACACGGCGATGTACTACTG 420 EK144 VH1-18 GAGCTGAGGAGCCTGAGATCTGACGACACGGCGGTGTACTACTG 421 EK137 CH1 Rev. GTAGGACAGCCGGGAAGG 422 Primer

Example 10.4 Homologous Recombination of PCR-Amplified CDRH3 Regions into Heavy Chain Chassis

After amplification, reaction products were pooled according to the respective VH chassis that they would ultimately be cloned into. Table 42 enumerates these pools, with the PCR primers used to obtain the CDRH3 sequences in each pool provided in the last two columns.

TABLE 42 PCR Primers Used to Amplify CDRH3 Regions from 424 Vectors Pool # (Arbitrary) HC Chassis Target 5′ Primer 3′ Primer 1 1-46 EK140 EK137 1-69 EK140 EK137 2 1-2 EK141 EK137 3 1-18 EK144 EK137 4 4-B EK139 EK137 4-31 EK139 EK137 4-34² EK139 EK137 4-39 EK139 EK137 4-59 EK139 EK137 4-61 EK139 EK137 5 5-51 EK143 EK137 6 3-15¹ EK133 EK137 3-7 EK133 EK137 3-33 EK133 EK137 3-33 EK133 EK137 3-48 EK133 EK137 7 3-23 EMK135 EK137 8 3-23* EK133 EK137 *Allowed the amplicons to be cloned into the other 5 VH3 family member chassis (i.e., other than VH3-23), while making the last three amino acids of these chassis CAK instead of the original CAR. ¹As described in Table 5, the original KT sequence in VH3-15 was mutated to RA, and the original TT to AR. ²As described in Table 5, the potential site for N-linked glycosylation was removed from CDRH2 of this chassis.

After pooling of the amplified CDRH3 regions, according to the process outlined above, the heavy chain chassis expression vectors were pooled according to their origin and cut, to create a “gap” for homologous recombination with the amplified CDRH3s. FIG. 6 shows a schematic structure of a heavy chain vector, prior to recombination with a CDRH3. In this exemplary embodiment of the invention, there were a total of 152 vectors encoding heavy chain chassis and IgG1 constant regions, but no CDRH3. These 152 vectors represent 17 individual variable heavy chain gene families (Table 5; Examples 1 and 2). Fifteen of the families were represented by the heavy chain chassis sequences described in Table 5 and the CDRH1/H2 variants described in Table 8 (i.e., 150 vectors). VH 3-30 differs from VH3-33 by a single amino acid; thus VH3-30 was included in the VH3-33 pool of variants. The 4-34 VH family member was kept separate from all others and, in this exemplary embodiment, no variants of it were included in the library. Thus, a total of 16 pools, representing 17 heavy chain chassis, were generated from the 152 vectors.

The vector pools were digested with the restriction enzyme SfiI, which cuts at two sites in the vector that are located between the end of the FRM3 of the variable domain and the start of the CH1 (SEQ ID NO: 573).

                                     VH3-48 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~   S  V  K   G  R  F  T   I  S  R   D  N  A   K  N  S  L   Y  L  Q   M  N  S   L  R  A  E • 2801 CTCTGTGAAG GGCCGATTCA CCATCTCCAG AGACAATGCC AAGAACTCAC TGTATCTGCA AATGAACAGC CTGAGAGCTG GAGACACTTC CCGGCTAAGT GGTAGAGGTC TCTGTTACGG TTCTTGAGTG ACATAGACGT TTACTTGTCG GACTCTCGAC       Constant DIAVYYCAR   ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ VH3-48                                                                       VTVSS common to all J ~~                                                                                ~~~~~                                    SfiI                                 SfiI                                ~~~~~~~~~~~~~~~                      ~~~~~~~~~~~~~~ •  D  T  A   V  Y  Y   C  A  R                                                                                    V  T • 2881 AGGACACGGC GGTGTACTAC TGCGCCAGAG GCCAATAGGG CCAACTATAA CAGGGGTACC CCGGCCAATA AGGCCGTCAC TCCTGTGCCG CCACATGATG ACGCGGTCTC CGGTTATCCC GGTTGATATT GTCCCCATGG GGCCGGTTAT TCCGGCAGTG VTVSS common to all J ~~~~~~~~~~~                                          hIgG1m17, 1            ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~             NheI            ~~~~~~ • V  S  S   A  S  T  K   G  P  S   V  F  P   L  A  P  S   S  K  S   T  S  G   G  T  A 2961 CGTCTCCTCA GCTAGCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG GCAGAGGAGT CGATCGTGGT TCCCGGGTAG CCAGAAGGGG GACCGTGGGA GGAGGTTCTC GTGGAGACCC CCGTGTCGCC

The gapped vector pools were then mixed with the appropriate (i.e., compatible) pool of CDRH3 amplicons, generated as described above, at a 50:1 insert to vector ratio. The mixture was then transformed into electrocompetent yeast (S. cerevisiae), which already contained plasmids or integrated genes comprising a VK light chain library (described below). The degree of library diversity was determined by plating a dilution of the electroporated cells on a selectable agar plate. In this exemplified embodiment of the invention, the agar plate lacked tryptophan and the yeast lacked the ability to endogenously synthesize tryptophan. This deficiency was remedied by the inclusion of the TRP marker on the heavy chain chassis plasmid, so that any yeast receiving the plasmid and recombining it with a CDRH3 insert would grow. The electroporated cells were then outgrown approximately 100-fold, in liquid media lacking tryptophan. Aliquots of the library were frozen in 50% glycerol and stored at −80° C. Each transformant obtained at this stage represents a clone that can express a full IgG molecule. A schematic diagram of a CDRH3 integrated into a heavy chain vector and the accompanying sequence are provided in FIG. 5.

A heavy chain library pool was then produced, based on the approximate representation of the heavy chain family members as depicted in Table 43.

TABLE 43 Occurrence of Heavy Chain Chassis in Data Sets Used to Design Library, Expected (Designed) Library, and Actual (Observed) Library Relative Occurrence in Chassis Data Sets (1) Expected (2) Observed (3) VH1-2 5.1 6.0 6.4 VH1-18 3.4 3.7 3.8 VH1-46 3.4 5.2 4.7 VH1-69 8.0 8.0 10.7 VH3-7 3.6 6.1 4.5 VH3-15 1.9 6.9 3.6 VH3-23 11.0 13.2 17.1 VH3-33/30 13.1 12.5 6.6 VH3-48 2.9 6.3 7.5 VH4-31 3.4 2.5 4.3 VH4-34 17.2 7.0 4.7 VH4-39 8.7 3.9 3.0 VH4-59 7.0 7.8 9.2 VH4-61 3.2 1.9 2.4 VH4-B 1.0 1.4 0.8 VH5-51 7.2 7.7 10.5 (1) As detailed in Example 1, these 17 sequences account for about 76% of the entire sample of human VH sequences used to represent the human repertoire. (2) Based on pooling of sub-libraries of each chassis type. (3) Usage in 531 sequences from library; cf. FIG. 20.

Example 10.5 K94R Mutation in VH3-23 and R94K Mutation in VH3-33, VH3-30, VH3-7, and VH3-48

This example describes the mutation of position 94 in VH3-23, VH3-33, VH3-30, VH3-7, and VH3-48. In VH3-23, the amino acid at this position was mutated from K to R. In VH3-33, VH3-30, VH3-7, and VH3-48, this amino acid was mutated from R to K. In VH3-32, this position was mutated from K to R. The purpose of making these mutations was to enhance the diversity of CDRH3 presentation in the library. For example, in naturally occurring VH3-23 sequences, about 90% have K at position 94, while about 10% have position R. By making these changes the diversity of the CDRH3 presentation is increased, as is the overall diversity of the library. ***

Amplification was performed using the 424 vectors as a template. For the K94R mutation, the vectors containing the sequence DTAVYYCAK (VH3-23; SEQ ID NO: 578) were amplified with a PCR primer that changed the K to a R and added 5′ tail for homologous recombination with the VH3-48, VH3-33, VH-30, and VH3-7. The “T” base in 3-48 does not change the amino acid encoded and thus the same primer with a T::C mismatch still allows homologous recombination into the 3-48 chassis.

Furthermore, the amplification products from the 424 vectors (produced as described above) containing the DTAVYYCAR (SEQ ID NO: 579) sequence can be homologously recombined into the VH3-23 (CAR) vector, changing R to K in this framework and thus further increasing the diversity of CDRH3 presentation in this chassis.

240                                                   294 VH3-48 (240) TCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCGGTGTACTACTGCGCCAGA SEQ ID NO: (574) VH3-33/30 (240) TCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAGA SEQ ID NO: (575) VH3-7 (240) TCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAGA SEQ ID NO: (576) VH3-23 (240) TCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAG SEQ ID NO: (577)

Example 11 VK Library Construction

This example describes the construction of a VK library of the invention. The exemplary VK library described herein corresponds to the VKCDR3 library of about 10⁵ complexity, described in Example 6.2. As described in Example 6, and throughout the application, other VK libraries are within the scope of the invention, as are Vλ libraries.

Ten VK chassis were synthesized (Table 11), which did not contain VKCDR3, but instead had two SfiI restriction sites in the place of VKCDR3, as for the heavy chain vectors. The kappa constant region followed the SfiI restriction sites. FIG. 8 shows a schematic structure of a light chain vector, prior to recombination with a CDRL3.

Ten VKCDR oligonucleotide libraries were then synthesized, as described in Example 6.2, using degenerate oligonucleotides (Table 33). The oligonucleotides were then PCR amplified, as separate pools, to make them double stranded and to add additional nucleotides required for efficient homologous recombination with the gapped (by SfiI) vector containing the VK chassis and constant region sequences. The VKCDR3 pools in this embodiment of the invention represented lengths 8, 9, and 10 amino acids, which were mixed post-PCR at a ratio 1:8:1. The pools were then cloned into the respective SfiI gapped VK chassis via homologous recombination, as described for the CDRH3 regions, set forth above. A schematic diagram of a CDRL3 integrated into a light chain vector and the accompanying sequence are provided in FIG. 9.

A kappa light chain library pool was then produced, based on the approximate representation of the VK family members found in the circulating pool of B cells. The 10 kappa variable regions used and the relative frequency in the final library pool are shown in Table 44.

TABLE 44 Occurrence of VK Chassis in Data Sets Used to Design Library, Expected (Designed) Library, and Actual (Observed) Library Relative Occurrence in Chassis Data Sets (1) Expected (2) Observed (3) VK1-5 8.6 7.1 5.8 VK1-12 4.0 3.6 3.5 VK1-27 3.3 3.6 8.1 VK1-33 5.3 7.1 3.5 VK1-39 18.5 21.4 17.4 VK2-28 7.7 7.1 5.8 VK3-11 10.9 10.7 20.9 VK3-15 6.6 7.1 4.7 VK3-20 24.5 21.4 18.6 VK4-1 10.4 10.7 11.6 (1) As indicated in Example 3, these 10 chassis account for about 80% of the occurrences in the entire data set of VK sequences examined. (2) Rounded off ratios from the data in column 2, then normalized for actual experimental set up. The relative rounded ratios are 6 for VK1-39 and VK3-20, 3 for VK3-11 and VK4-1, 2 for VK-15, VK1-33, VK2-28 and VK3-15, and 1 for VK1-12 and VK1-27. (3) Chassis usage in set of 86 sequences obtained from library; see also FIG. 22.

Example 12 Characterization of Exemplary Libraries

This example shows the characteristics of exemplary libraries of the invention, constructed according to the methods described herein.

Example 12.1 Characterization of the Heavy Chains

To characterize the product of the split pool synthesis, ten of the 424 vectors containing the [Tail]-[N1]-[DH]-[N2]-[H3-JH] product were selected at random and transformed into E. coli. The split pool product had a theoretical diversity of about 1.1×10⁶ (i.e., 278×141×28). Ninety-six colonies were selected from the transformation and forward and reverse sequences were generated for each clone. Of the 96 sequencing reactions, 90 yielded sequences from which the CDRH3 region could be identified, and about 70% of these sequences matched a designed sequence in the library. The length distribution of the sequenced CDRH3 segments from the ten vectors, as compared to the theoretical distribution (based on design), is provided in FIG. 10. The length distribution of the individual DH, N2, and H3-JH segments obtained from the ten vectors are shown in FIGS. 11-13.

Once the length distribution of the CDRH3 components of the library that were contained in the vector matched design were verified, the CDRH3 domains and heavy chain family representation in yeast that had been transformed according to the process described in Example 10.4 were characterized. Over 500 single-pass sequences were obtained. Of these, 531 yielded enough sequence information to identify the heavy chain chassis and 291 yielded enough sequence information to characterize the CDRH3. These CDRH3 domains have been integrated with the heavy chain chassis and constant region, according to the homologous recombination processes described herein. The length distribution of the CDRH3 domains from 291 sequences, compared to the theoretical length distribution, is shown in FIG. 14. The mean theoretical length was 14.4±4 amino acids, while the average observed length was 14.3±3 amino acids. The observed length of each portion of the CDRH3, as compared to theoretical, is presented in FIGS. 15-18. FIG. 19 depicts the familial origin of the JH segments identified in the 291 sequences, and FIG. 20 shows the representation of 16 of the chassis of the library. The VH3-15 chassis was not represented amongst these sequences. This was corrected later by introducing yeast transformants containing the VH3-15 chassis, with CDRH3 diversity, into the library at the desired composition.

Example 12.2 Characterization of the Light Chains

The length distribution of the CDRL3 components, from the VKCDR3 library described in Example 6.2, were determined after yeast transformation via the methods described in Example 10.4. A comparison of the CDRL3 length from 86 sequences of the library to the human sequences and designed sequences is provided in FIG. 21. FIG. 22 shows the representation of the light chain chassis from amongst the 86 sequences selected from the library. About 91% of the CDRL3 sequences were exact matches to the design, and about 9% differed by a single amino acid.

Example 13 Characterization of the Composition of the Designed CDRH3 Libraries

This example presents data on the composition of the CDRH3 domains of exemplary libraries, and a comparison to other libraries of the art. More specifically, this example presents an analysis of the occurrence of the 400 possible amino acid pairs (20 amino acids×20 amino acids) occurring in the CDRH3 domains of the libraries. The prevalence of these pairs is computed by examination of the nearest neighbor (i-i+1; designated IP1), next nearest neighbor (1-i+2; designated IP2), and next-next nearest neighbor (1-i+3; designated IP3) of the i residue in CDRH3. Libraries previously known in the art (e.g., Knappik et al., J. Mol. Biol., 2000, 296: 57; Sidhu et al., J. Mol. Biol., 2004, 338: 299; and Lee et al., J. Mol. Biol. 2004, 340: 1073, each of which is incorporated by reference in its entirety) have only considered the occurrence of the 20 amino acids at individual positions within CDRH3, while maintaining the same composition across the center of CDRH3, and not the pair-wise occurrences considered herein. In fact, according to Sidhu et al. (J. Mol. Biol., 2004, 338: 299, incorporated by reference in its entirety), “[i]n CDR-H3, there was some bias towards certain residue types, but all 20 natural amino acid residues occurred to a significant extent, and there was very little position-specific bias within the central portion of the loop”. Thus, the present invention represents the first recognition that, surprisingly, a position-specific bias does exist within the central portion of the CDRH3 loop, when the occurrences of amino acid pairs recited above are considered. This example shows that the libraries described herein more faithfully reproduce the occurrence of these pairs as found in human sequences, in comparison to other libraries of the art. The composition of the libraries described herein may thus be considered more “human” than other libraries of the art.

To examine the pair-wise composition of CDRH3 domains, a portion of CDRH3 beginning at position 95 was chosen. For the purposes of comparison with data presented in Knappik et al. and Lee et al., the last five residues in each of the analyzed CDRH3s were ignored. Thus, for the purposes of this analysis, both members of the pair i-i+X (X=1 to 3) must fall within the region starting at position 95 and ending at (but including) the sixth residue from the C-terminus of the CDRH3. The analyzed portion is termed the “central loop” (see Definitions).

To estimate pair distributions in representative libraries of the invention, a sampling approach was used. A number of sequences were generated by choosing randomly and, in turn, one of the 424 tail plus N1 segments, one of the 278 DH segments, one of the 141 N2 segments and one of the 28 JH segments (the latter truncated to include only the 95 to 102 Kabat CDRH3). The process was repeated 10,000 times to generate a sample of 10,000 sequences. By choosing a different seed for the random number generation, an independent sample of another 10,000 sequences was also generated and the results for pair distributions were observed to be nearly the same. For the calculations presented herein, a third and much larger sample of 50,000 sequences was used. A similar approach was used for the alternative library embodiment (N-1-141), whereby the first segment was selected from 1068 tail+N1 segments (resulting after eliminating redundant sequences from 2 times 4 times 141 or 1128 possible combinations).

The pair-wise composition of Knappik et al. was determined based on the percent occurrences presented in FIG. 7a of Knappik et al. (p. 71). The relevant data are reproduced below, in Table 45.

TABLE 45 Composition of CDRH3 positions 95-100s (corresponding to positions 95-99B of the libraries of the current invention) of CDRH3 of Knappik et al. (from FIG. 7a of Knappik et al.) Amino Acid Planned (%) Found (%) A 4.1 3.0 C 1.0 1.0 D 4.1 4.2 E 4.1 2.3 F 4.1 4.9 G 15.0 10.8 H 4.1 4.6 I 4.1 4.5 K 4.1 2.9 L 4.1 6.6 M 4.1 3.3 N 4.1 4.5 P 4.1 4.8 Q 4.1 2.9 R 4.1 4.1 S 4.1 5.6 T 4.1 4.5 V 4.1 3.7 W 4.1 2.0 Y 15.0 19.8

The pair-wise composition of Lee et al. was determined based on the libraries depicted in Table 5 of Lee et al., where the positions corresponding to those CDRH3 regions analyzed from the current invention and from Knappik et al. are composed of an “XYZ” codon in Lee et al. The XYZ codon of Lee et al. is a degenerate codon with the following base compositions:

position 1 (X): 19% A, 17% C, 38% G, and 26% T;

position 2 (Y): 34% A, 18% C, 31% G, and 17% T; and

position 3 (Z): 24% G and 76% T.

When the approximately 2% of codons encoding stop codons are excluded (these do not occur in functionally expressed human CDRH3 sequences), and the percentages are re-normalized to 100%, the following amino acid representation can be deduced from the composition of the XYZ codon of Lee et al. (Table 46).

TABLE 46 Composition of CDRH3 of Lee et al., Based on the Composition of the Degenerate XYZ Codon. Type Percent A 6.99% C 6.26% D 10.03% E 3.17% F 3.43% G 12.04% H 4.49% I 2.51% K 1.58% L 4.04% M 0.79% N 5.02% P 3.13% Q 1.42% R 6.83% S 9.35% T 3.49% V 6.60% W 1.98% Y 6.86%

The occurrences of each of the 400 amino acid pairs, in each of the IP1, IP2, and IP3 configurations, can be computed for Knappik et al. and Lee et al. by multiplying together the individual amino acid compositions. For example, for Knappik et al., the occurrence of YS pairs in the library is calculated by multiplying 15% by 4.1%, to yield 6.1%; note that the occurrence of SY pairs would be the same. Similarly, for the XYZ codon-based libraries of Lee et al., the occurrence of YS pairs would be 6.86% (Y) multiplied by 9.35% (S), to give 6.4%; the same, again, for SY.

For the human CDRH3 sequences, the calculation is performed by ignoring the last five amino acids in the Kabat definition. By ignoring the C-terminal 5 amino acids of the human CDRH3, these sequences may be compared to those of Lee et al., based on the XYZ codons. While Lee et al. also present libraries with “NNK” and “NNS” codons, the pair-wise compositions of these libraries are even further away from human CDRH3 pair-wise composition. The XYZ codon was designed by Lee et al. to replicate, to some extent, the individual amino acid type biases observed in CDRH3.

An identical approach was used for the libraries of the invention, after using the methods described above to produce sample sequences. While it is possible to perform these calculations with all sequences in the library, independent random samples of 10,000 to 20,000 members gave indistinguishable results. The numbers reported herein were thus generated from samples of 50,000 members.

Three tables were generated for IP1, IP2 and IP3, respectively (Tables 47, 48, and 49). Out of the 400 pairs, a selection from amongst the 20 most frequently occurring is included in the tables. The sample of about 1,000 human sequences (Lee et al., 2006) is denoted as “Preimmune,” a sample of about 2,500 sequences (Jackson et al., 2007) is denoted as “Humabs,” and the more affinity matured subset of the latter, which excludes all of the Preimmune set, is denoted as “Matured.” Synthetic libraries in the art are denoted as HuCAL (Knappik, et al., 2000) and XYZ (Lee et al., e 2004). Two representative libraries of the invention are included: LUA-59 includes 59 N1 segments, 278 DH segments, 141 N2 segments, and 28 H3-JH segments (see Examples, above). LUA-141 includes 141 N1 segments, 278 DH segments, 141 N2 segments, and 28 H3-JH segments (see Examples, above). Redundancies created by combination of the N1 and tail sequences were removed from the dataset in each respective library. In certain embodiments, the invention may be defined based on the percent occurrence of any of the 400 amino acid pairs, particularly those in Tables 47-49. In certain embodiments, the invention may be defined based on at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of these pairs. In certain embodiments of the invention, the percent occurrence of certain pairs of amino acids may fall within ranges indicated by “LUA-” (lower boundary) and “LUA+” (higher boundary), in the following tables. In some embodiments of the invention, the lower boundary for the percent occurrence of any amino acid pairs may be about 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, and 5. In some embodiments of the invention, the higher boundary for the percent occurrence of any amino acid pairs may be about 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, and 8. According to the present invention, any of the lower boundaries recited may be combined with any of the higher boundaries recited, to establish ranges, and vice-versa.

TABLE 47 Percent Occurrence of i − i + 1 (IP1) Amino Acid Pairs in Human Sequences, Exemplary Libraries of the Invention, and the Libraries of Knappik et al. and Lee et al. LUA- LUA- Pairs Preimmune Humabs Matured 59 141 HuCAL XYZ LUA− LUA+ Range HuCAL XYZ YY 5.87 4.44 3.27 5.83 5.93 2.25 0.47 2.50 6.50 4.00 0 0 SG 3.54 3.41 3.26 3.90 3.72 0.61 1.13 2.50 4.50 2.00 0 0 SS 3.35 2.65 2.26 2.82 3.08 0.16 0.88 2.00 4.00 2.00 0 0 GS 2.59 2.37 2.20 3.82 3.52 0.61 1.13 1.50 4.00 2.50 0 0 GY 2.55 2.34 2.12 3.15 2.56 2.25 0.83 2.00 3.50 1.50 1 0 GG 2.19 2.28 2.41 6.78 3.51 2.25 1.45 2.00 7.00 5.00 1 0 YS 1.45 1.30 1.23 1.40 1.52 0.61 0.64 0.75 2.00 1.25 0 0 YG 1.35 1.21 1.10 1.64 1.69 2.25 0.83 0.75 2.00 1.25 0 1 SY 1.31 1.07 0.90 1.65 1.77 0.61 0.64 0.75 2.00 1.25 0 0 YD 1.67 1.40 1.17 0.88 0.90 0.61 0.69 0.75 2.25 1.50 0 0 DS 1.53 1.31 1.16 1.20 1.46 0.16 0.94 0.75 2.00 1.25 0 1 DY 1.40 1.23 1.11 0.34 0.48 0.61 0.69 0.25 2.00 1.75 1 1 VV 1.37 0.94 0.64 2.30 2.30 0.16 0.44 0.50 2.50 2.00 0 0 GD 1.20 1.21 1.25 0.49 0.44 0.61 1.21 0.25 1.75 1.50 1 1 AA 1.16 0.93 0.75 1.27 1.46 0.16 0.49 0.60 1.50 0.90 0 0 RG 1.08 1.26 1.38 1.69 1.38 0.61 0.82 1.00 2.00 1.00 0 0 VA 0.91 0.66 0.46 0.36 0.35 0.16 0.46 0.25 1.00 0.75 0 1 GV 0.84 0.89 0.95 2.87 2.16 0.61 0.79 0.80 3.00 2.20 0 0 CS 0.82 0.55 0.38 0.79 0.80 0.04 0.59 0.50 1.00 0.50 0 1 GR 0.74 0.90 1.00 1.01 0.79 0.61 0.82 0.70 1.25 0.55 0 1 The pairs in bold comprise about 19% to about 24% of occurrences (among the possible 400 pairs) for the Preimmune (Lee, et al., 2006), Humabs (Jackson, et al., 2007) and matured (Jackson minus Lee) sets. They account for about 27% to about 31% of the occurrences in the LUA libraries, but only about 12% in the HuCAL library and about 8% in the “XYZ” library. This is a reflection of the fact that pair-wise biases do exist in the human and LUA libraries, but not in the others. The last 2 columns indicate whether the corresponding pair-wise compositions fall within the LUA− and LUA+ boundaries: 0 if outside, 1 if within.

TABLE 48 Percent Occurrence of i − i + 2 (IP2) Amino Acid Pairs in Human Sequences, Exemplary Libraries of the Invention, and the Libraries of Knappik et al. and Lee et al. LUA- LUA- Pairs Preimmune Humabs Matured 59 141 HuCAL XYZ LUA− LUA+ Range HuCAL XYZ YY 3.57 2.59 1.78 2.99 3.11 2.25 0.47 2.5 4.5 2 0 0 GY 3.34 2.91 2.56 4.96 3.78 2.25 0.83 2.5 5.5 3 0 0 SY 2.94 2.41 2.01 3.03 3.42 0.61 0.64 2 4 2 0 0 YS 2.88 2.34 1.95 3.24 3.32 0.61 0.64 1.75 3.75 2 0 0 SG 2.60 2.29 2.05 2.84 2.96 0.61 1.13 2 3.5 1.5 0 0 SS 2.27 2.01 1.84 2.30 2.50 0.16 0.88 1.5 3 1.5 0 0 GS 2.16 2.12 2.10 2.96 2.32 0.61 1.13 1.5 3 1.5 0 0 GG 1.92 2.25 2.44 6.23 3.68 2.25 1.45 1.5 7 5.5 1 0 YG 1.17 1.14 1.15 1.39 1.47 2.25 0.83 1 2 1 0 0 DS 2.03 1.67 1.40 1.21 1.48 0.16 0.94 1 2.5 1.5 0 0 YD 1.71 1.39 1.11 0.89 0.92 0.61 0.69 0.75 1.75 1 0 0 VG 1.35 1.17 1.01 1.75 1.54 0.61 0.79 1 2 1 0 0 DY 1.06 1.02 0.99 0.23 0.40 0.61 0.69 0.2 1.2 1 1 1 WG 1.06 0.76 0.53 0.85 0.91 0.61 0.24 0.75 1.25 0.5 0 0 RY 0.98 1.00 0.96 0.70 0.91 0.61 0.47 0.6 1 0.4 1 0 GC 0.97 0.75 0.64 0.94 0.81 0.15 0.75 0.5 1 0.5 0 1 DG 0.95 1.05 1.08 1.78 1.05 0.61 1.21 0.75 2 1.25 0 1 GD 0.94 0.88 0.86 0.47 0.36 0.61 1.21 0.25 1 0.75 1 0 VV 0.94 0.59 0.35 0.95 0.90 0.16 0.44 0.5 1 0.5 0 0 AA 0.90 0.73 0.59 0.72 0.74 0.16 0.49 0.5 1 0.5 0 0 The pairs in bold comprise about 18% to about 23% of occurrences (among the possible 400 pairs) for the Preimmune (Lee, et al., 2006), Humabs (Jackson, et al., 2007) and matured (Jackson minus Lee) sets. They account for about 27% to about 30% of the occurrences in the LUA libraries, but only about 12% in the HuCAL library and about 8% in the “XYZ” library. Because of the nature of the construction of the central loops in the HuCAL and XYZ libraries, these numbers are the same for the IP1, IP2, and IP3 pairs. The last 2 columns indicate whether the corresponding pair-wise compositions fall within the LUA− and LUA+ boundaries: 0 if outside, 1 if within.

TABLE 49 Percent Occurrence of i − i + 3 (IP3) Amino Acid Pairs in Human Sequences, Exemplary Libraries of the Invention, and the Libraries of Knappik et al. and Lee et al. LUA- LUA- Pairs Preimmune Humabs Matured 59 141 HuCAL XYZ LUA− LUA+ Range HuCAL XYZ GY 3.55 2.85 2.32 5.80 4.42 2.25 0.83 2.5 6.5 4 0 0 SY 3.38 3.01 2.67 3.78 4.21 0.61 0.64 1 5 4 0 0 YS 3.18 2.56 2.05 3.20 3.33 0.61 0.64 2 4 2 0 0 SS 2.26 1.74 1.37 1.81 2.18 0.16 0.88 1 3 2 0 0 GS 2.23 2.13 2.00 4.60 3.33 0.61 1.13 2 5 3 0 0 YG 2.14 1.65 1.35 2.69 2.79 2.25 0.83 1.5 3 1.5 1 0 YY 1.86 1.48 1.12 1.18 1.27 2.25 0.47 0.75 2 1.25 0 0 GG 1.60 1.87 2.11 4.73 2.84 2.25 1.45 1.5 5 3.5 1 0 SG 0.90 1.04 1.12 0.93 1.25 0.61 1.13 0.75 1.5 0.75 0 1 DG 2.01 1.94 1.84 2.51 2.03 0.61 1.21 1.5 3 1.5 0 0 DS 1.48 1.31 1.22 0.41 0.55 0.16 0.94 0.25 1.5 1.25 0 1 VA 1.18 0.83 0.55 1.48 1.46 0.16 0.46 0.5 2 1.5 0 0 AG 1.13 1.09 1.03 0.97 1.04 0.61 0.84 0.9 2 1.1 0 0 TY 1.05 0.90 0.76 1.01 1.16 0.61 0.24 0.75 1.75 1 0 0 PY 1.02 0.88 0.79 1.23 0.86 0.61 0.21 0.75 1.75 1 0 0 RS 1.02 0.88 0.77 0.38 0.55 0.16 0.64 0.25 1.25 1 0 1 RY 1.02 1.12 1.14 0.68 0.88 0.61 0.47 0.65 1.25 0.6 0 0 LY 1.01 0.88 0.75 0.69 0.76 0.61 0.28 0.65 1.25 0.6 0 0 DY 0.93 0.84 0.77 0.72 0.95 0.61 0.69 0.7 1.3 0.6 0 0 GC 0.90 0.62 0.48 0.86 0.68 0.15 0.75 0.5 1 0.5 0 1 The pairs in bold make up about 16 to about 21% of the occurrences (among the possible 400 pairs) for the Preimmune (Lee, et al., 2006), Humabs (Jackson, et al., 2007) and matured (Jackson minus Lee) sets. They account for 26 to 29% of the occurrences in the LUA libraris, but only about 12% in the HuCAL library and about 8% for the “XYZ” library. Because of the nature of the construction of the central loops in the HuCAL and XYZ libraries, these numbers are the same for the IP1, IP2, and IP3 pairs. The last 2 columns indicate whether the corresponding pair-wise compositions fall within the LUA− and LUA+ boundaries: 0 if outside, 1 if within.

The analysis provided in this example demonstrates that the composition of the libraries of the present invention more closely mimics the composition of human sequences than other libraries known in the art. Synthetic libraries of the art do not intrinsically reproduce the composition of the “central loop” portion actual human CDRH3 sequences at the level of pair percentages. The libraries of the invention have a more complex pair-wise composition that closely reproduces that observed in actual human CDRH3 sequences. The exact degree of this reproduction versus a target set of actual human CDRH3 sequences may be optimized, for example, by varying the compositions of the segments used to design the CDRH3 libraries. Moreover, it is also possible to utilize these metrics to computationally design libraries that exactly mimic the pair-wise compositional prevalence found in human sequences.

Example 14 Information Content of Exemplary Libraries

One way to quantify the observation that certain libraries, or collection of sequences, may be intrinsically more complex or “less random” than others is to apply information theory (Shannon, Bell Sys. Tech. J., 1984, 27: 379; Martin et al., Bioinformatics, 2005, 21: 4116; Weiss et al., J. Theor. Biol., 2000, 206: 379, each incorporated by reference in its entirety). For example, a metric can be devised to quantify the fact that a position with a fixed amino acid represents less “randomness” than a position where all 20 amino acids may occur with equal probability. Intermediate situations should lead, in turn, to intermediate values of such a metric. According to information theory this metric can be represented by the formula:

$I = {\sum\limits_{i = 1}^{N}\; {f_{i}\mspace{11mu} \log_{2}\mspace{11mu} f_{i}}}$

Here, f_(i) is the normalized frequency of occurrence of i, which may be an amino acid type (in which case N would be equal to 20). When all f_(i) are zero except for one, the value of I is zero. In any other case the value of I would be smaller, i.e., negative, and the lowest value is achieved when all f_(i) values are the same and equal to N. For the amino acid case, N is 20, and the resulting value of I would be −4.322. Because I is defined with base 2 logarithms, the units of I are bits.

The I value for the HuCAL and XYZ libraries at the single position level may be derived from Tables 45 and 46, respectively, and are equal to −4.08 and −4.06. The corresponding single residue frequency occurrences in the non-limiting exemplary libraries of the invention and the sets of human sequences previously introduced, taken within the “central loop” as defined above, are provided in Table 50.

TABLE 50 Amino Acid Type Frequencies in Central Loop Type Preimmune Humabs Matured LUA-59 LUA-141 A 5.46 5.51 5.39 5.71 6.06 C 1.88 1.46 1.22 1.33 1.34 D 7.70 7.51 7.38 4.76 5.23 E 2.40 2.90 3.28 3.99 4.68 F 2.29 2.60 2.81 1.76 2.17 G 14.86 15.42 15.82 24.90 18.85 H 1.46 1.79 2.01 0.20 0.67 I 3.71 3.26 2.99 3.99 4.34 K 1.06 1.27 1.44 0.21 0.67 L 4.48 4.84 5.16 4.12 4.54 M 1.18 1.03 0.93 0.94 1.03 N 1.81 2.43 2.84 0.41 0.65 P 4.12 4.10 4.13 5.68 3.96 Q 1.60 1.77 1.95 0.21 0.68 R 5.05 5.90 6.41 3.35 4.11 S 12.61 11.83 11.37 11.18 12.77 T 4.59 5.11 5.47 4.36 4.95 V 6.21 5.55 5.12 8.13 7.67 W 2.79 2.91 3.07 1.57 1.98 Y 14.74 12.81 11.24 13.20 13.63 The information content of these sets, computed by the formula given above, would then be −3.88, −3.93, −3.96, −3.56, and −3.75, for the preimmune, human, matured, LUA-59 and LUA-141 sets, respectively. As the frequencies deviate more from completely uniform (5% for each of the 20), then numbers tend to be larger, or less negative.

The identical approach can be used to analyze pair compositions, or frequencies, by calculating the sum in the formula above over the 20×20 or 400 values of the frequencies for each of the pairs. It can be shown that any pair frequency made up of the simple product of two singleton frequency sets is equal to the sum of the individual singleton I values. If the two singleton frequency sets are the same or approximately so, this means that I (independent pairs)=2*I (singles). It is thus possible to define a special case of the mutual information, MI, for a general set of pair frequencies as MI (pair)=I(pair)−2*I (singles) to measure the amount of information gained by the structure of the pair frequencies themselves (compare to the standard definitions in Martin et al., 2005, for example, after considering that I(X)=−H(X) in their notation). When there is no such structure, the value of MI is simply zero.

Values of MI computed from the pair distributions discussed above (over the entire set of 400 values) are given in Table 51.

TABLE 51 Mutual Information Within Central Loop of CDRH3 Library or Set i - i + 1 i - i + 2 i - i + 3 Preimmune 0.226 0.192 0.163 Humabs 0.153 0.128 0.111 Matured 0.124 0.107 0.100 LUA-59 0.422 0.327 0.278 LUA-141 0.376 0.305 0.277 HuCAL 0.000 0.000 0.000 XYZ 0.000 0.000 0.000

It is notable that the MI values decrease within sets of human sequences as those sequences undergo further somatic mutation, a process that over many independent sequences is essentially random. It is also worth noting that the MI values decrease as the pairs being considered sit further and further apart, and this is the case for both sets of human sequences, and exemplary libraries of the invention. In both cases, as the two amino acids in a pair become further separated the odds of their straddling an actual segment (V, D, J plus V-D or D-J insertions) increase, and their pair frequencies become closer to a simple product of singleton frequencies.

Table 52 contains sequence information on certain immunoglobulin gene segments cited in the application. These sequences are non-limiting, and it is recognized that allelic variants exist and encompassed by the present invention. Accordingly, the methods present herein can be utilized with mutants of these sequences.

TABLE 52 Sequence Information for Certain Immunoglobulin Gene Segments Cited Herein SEQ ID NO: Sequence Peptide or Nucleotide Sequence Observations 423 IGHV1-3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQ APGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSAST AYMELSSLRSEDTAVYYCAR 424 IGHV1-8_v1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQ ATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTR NTS IS TAYMELSSLRSEDTAVYYCAR 425 IGHV1-8_v2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQ N to D mutation avoids NTS ATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTR DTS IS potential glycosylation site in the TAYMELSSLRSEDTAVYYCAR original germline sequence (v1 above). XTS, where X is not N, and NTZ, where Z is not S or T are also options. NPS is yet another option that is much less likely to be N-linked glycosylated. 426 IGHV1-24 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQ APGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDT AYMELSSLRSEDTAVYYCAT 427 IGHV1-45 QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYRYLHWVRQ APGQALEWMGWITPFNGNTNYAQKFQDRVTITRDRSMST AYMELSSLRSEDTAMYYCAR 428 IGHV1-58 QMQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQ ARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTST AYMELSSLRSEDTAVYYCAA 429 IGHV2-5 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWI RQPPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTSKN QVVLTMTNMDPVDTATYYCAHR 430 IGHV2-26 QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWI RQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKS QVVLTMTNMDPVDTATYYCARI 431 IGHV2-70_v1 RVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGM C VSWI RQPPGKALEWLARIDWDDDKYYSTSLKTRLTISKDTSKN QVVLTMTNMDPVDTATYYCARI 432 IGHV2-70_v2 RVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGM G VSWI C to G mutation avoids unpaired RQPPGKALEWLARIDWDDDKYYSTSLKTRLTISKDTSKN Cys in v1 above. G was chosen QVVLTMTNMDPVDTATYYCARI by analogy to other germline sequences, but other amino acid types, R, S, T, as non-limiting examples, are possible. 433 IGHV3-9 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ APGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNS LYLQMNSLRAEDTALYYCAKD 434 IGHV3-11 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQ APGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCAR 435 IGHV3-13 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQ ATGKGLEWVSAIGTAGDTYYPGSVKGRFTISRENAKNSL YLQMNSLRAGDTAVYYCAR 436 IGHV3-20 EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVR QAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAK NSLYLQMNSLRAEDTALYHCAR 437 IGHV3-21 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVR QAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAK NSLYLQMNSLRAEDTAVYYCAR 438 IGHV3-43 EVQLVESGGWVQPGGSLRLSCAASGFTFDDYTMHWVRQ APGKGLEWVSLISWDGGSTYYADSVKGRFTISRDNSKN SLYLQMNSLRTEDTALYYCAKD 439 IGHV3-49 EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQ APGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSK SIAYLQMNSLKTEDTAVYYCTR 440 IGHV3-53 EVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMSWVRQ APGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAR 441 IGHV3-64 EVQLVESGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQ APGKGLEYVSAISSNGGSTYYADSVKGRFTISRDNSKNT LYLQMSSLRAEDTAVYYCVK 442 IGHV3-66 EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQ APGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAR 443 IGHV3-72 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQ APGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDDSK NSLYLQMNSLKTEDTAVYYCAR 444 IGHV3-73 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQ ASGKGLEWVGRIRSKANSYATAYAASVKGRFTISRDDSK NTAYLQMNSLKTEDTAVYYCTR 445 IGHV3-74 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQ APGKGLVWVSRINSDGSSTSYADSVKGRFTISRDNAKNT LYLQMNSLRAEDTAVYYCAR 446 IGHV4-4v1 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVR Contains CDRH1 with size 6 QPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQ (Kabat definition); canonical FSLKLSSVTAADTAVYYCAR structure H1-2. Sequence corresponds to allele *02 of IGHV4-4. 447 IGHV4-4v2 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQ Contains CDRH1 with size 5 PAGKGLEWIGRIYTSGSTNYNPSLKSRVTMSVDTSKNQF (Kabat definition); canonical SLKLSSVTAADTAVYYCAR structure H1-1. Sequence corresponds to allele *07 of IGHV4-4 448 IGHV4-28 QVQLQESGPGLVKPSDTLSLTCAVSGYSISSSNWWGWIR QPPGKGLEWIGYIYYSGSTYYNPSLKSRVTMSVDTSKNQ FSLKLSSVTAVDTAVYYCAR 449 IGHV6-1 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWI RQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTS KNQFSLQLNSVTPEDTAVYYCAR 450 IGHV7-4-1 QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQ APGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVST AYLQISSLKAEDTAVYYCAR 451 IGKV1-06 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQK PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCLQDYNYP 452 IGKV1-08_v1 AIRMTQSPSSFSASTGDRVTITCRASQGISSYLAWYQQK PGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTIS C L QSEDFATYYCQQYYSYP 453 IGKV1-08_v2 AIRMTQSPSSFSASTGDRVTITCRASQGISSYLAWYQQK C to S mutation avoids unpaired PGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTIS S L Cys. in v1 above. S was chosen QSEDFATYYCQQYYSYP by analogy to other germline sequences, but amino acid types, N, R, 5, as non-limiting examples, are also possible 454 IGKV1-09 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQK PGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSL QPEDFATYYCQQLNSYP 455 IGKV1-13 AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQK PGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQFNSYP 456 IGKV1-16 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQK PGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQYNSYP 457 IGKV1-17 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQK PGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSL QPEDFATYYCLQHNSYP 458 IGKV1-37_v1 DIQLTQSPSSLSASVGDRVTITCRVSQGISSYLNWYRQK PGKVPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSL QPEDVATYY G QRTYNAP 459 IGKV1-37_v2 DIQLTQSPSSLSASVGDRVTITCRVSQGISSYLNWYRQK Restores conserved Cys, missing PGKVPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSL in v1 above, just prior to CDRL3. QPEDVATYY C QRTYNAP 460 IGKV1D-16 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQK PEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQYNSYP 461 IGKV1D-17 NIQMTQSPSAMSASVGDRVTITCRARQGISNYLAWFQQK PGKVPKHLIYAASSLQSGVPSRFSGSGSGTEFTLTISSL QPEDFATYYCLQHNSYP 462 IGKV1D-43 AIRMTQSPFSLSASVGDRVTITCWASQGISSYLAWYQQK PAKAPKLFIYYASSLQSGVPSRFSGSGSGTDYTLTISSL QPEDFATYYCQQYYSTP 463 IGKV1D8_v1 VIWMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQK PGKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTIS C L QSEDFATYYCQQYYSFP 464 IGKV1D-8_v2 VIWMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQK C to S mutation avoids unpaired PGKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTIS S L Cys. in v1 above. S was chosen QSEDFATYYCQQYYSFP by analogy to other germline sequences, but amino acid types, N, R, 5, as non-limiting examples, are also possible 465 IGKV2-24 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLS WLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTL KISRVEAEDVGVYYCMQATQFP 466 IGKV2-29 DIVMTQTFLSLSVTRQQPASISCKSSQSLLHSDGVTYLY WYLQRPQQSPQLLTYEVSSRFSGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCMQGTHLP 467 IGKV2-30 DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLN WFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCMQGTHWP 468 IGKV2-40 DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYL DWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFT LKISRVEAEDVGVYYCMQRIEFP 469 IGKV2D-26 EIVMTQTPLSLSITPGEQASMSCRSSQSLLHSDGYTYLY WFLQKARPVSTLLIYEVSNRFSGVPDRFSGSGSGTDFTL KISRVEAEDFGVYYCMQDAQD 470 IGKV2D-29 DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLY WYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCMQSIQLP 471 IGKV2D-30 DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLN WFQQRPGQSPRRLIYKVSNWDSGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCMQGTHWP 472 IGKV3D-07 EIVMTQSPATLSLSPGERATLSCRASQSVSSSYLSWYQQ KPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISS LQPEDFAVYYCQQDYNLP 473 IGKV3D-11 EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSL EPEDFAVYYCQQRSNWH 474 IGKV3D-20 EIVLTQSPATLSLSPGERATLSCGASQSVSSSYLAWYQQ KPGLAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSSP 475 IGKV5-2_v1 ETTLTQSPAFMSATPGDKV NIS CKASQDIDDDMNWYQQK PGEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNI ESEDAAYYFCLQHDNFP 476 IGKV5-2_v2 ETTLTQSPAFMSATPGDKV TIS CKASQDIDDDMNWYQQK N to D mutation avoids NIS PGEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNI potential glycosylation site in v1 ESEDAAYYFCLQHDNFP above. XIS, where X is not N, and NIZ, where Z is not S or T are also options. NPS is yet another option that is much less likely to be N- linked glycosylated. 477 IGKV6-21 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQK PDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSL EAEDAATYYCHQSSSLP 478 IGKV6D-21 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQK PDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSL EAEDAATYYCHQSSSLP 479 IGKV7-3 DIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHW YQQKPGQPPKLLIYQASNKDTGVPARFSGSGSGTDFTLT INPVEANDTANYYCLQSKNFP 480 IGλV1-36 QSVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQ LPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISG LQSEDEADYYCAAWDDSLNG 481 IGλV1-47 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQ LPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISG LRSEDEADYYCAAWDDSLSG 482 IGλV10-54 QAGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQ HQGHPPKLLSYRNNNRPSGISERLSASRSGNTASLTITG LQPEDEADYYCSAWDSSLSA 483 IGλV2-11_v1 QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQ QHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTIS GLQAEDEADYYC C SYAGSYTF 484 IGλV2-11_v2 QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQ C to S mutation avoids unpaired QHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTIS Cys in v1 above. S was chosen GLQAEDEADYYC S SYAGSYTF by analogy to other germline sequences, but other amino acid types, such as Q, G, A, L, as non- limiting examples, are also possible 485 IGλV2-18 QSALTQPPSVSGSPGQSVTISCTGTSSDVGSYNRVSWYQ QPPGTAPKLMIYEVSNRPSGVPDRFSGSKSGNTASLTIS GLQAEDEADYYCSLYTSSSTF 486 IGλV2-23_v1 QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQ QHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTIS GLQAEDEADYYC C SYAGSSTL 487 IGλV2-23_v2 QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQ C to S mutation avoids unpaired QHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTIS Cys in v1 above. S was chosen by GLQAEDEADYYC S SYAGSSTL analogy to other germline sequences, but other amino acid types, such as Q, G, A, L, as non- limiting examples, are also possible 488 IGλV2-8 QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQ QHPGKAPKLMIYEVSKRPSGVPDRFSGSKSGNTASLTVS GLQAEDEADYYCSSYAGSNNF 489 IGλV3-10 SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKS GQAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTISGAQ VEDEADYYCYSTDSSGNH 490 IGλV3-12 SYELTQPHSVSVATAQMARITCGGNNIGSKAVHWYQQKP GQDPVLVIYSDSNRPSGIPERFSGSNPGNTTTLTISRIE AGDEADYYCQVWDSSSDH 491 IGλV3-16 SYELTQPPSVSVSLGQMARITCSGEALPKKYAYWYQQKP GQFPVLVIYKDSERPSGIPERFSGSSSGTIVTLTISGVQ AEDEADYYCLSADSSGTY 492 IGλV3-25 SYELMQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKP GQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQ AEDEADYYCQSADSSGTY 493 IGλV3-27 SYELTQPSSVSVSPGQTARITCSGDVLAKKYARWFQQKP GQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGAQ VEDEADYYCYSAADNN 494 IGλV3-9 SYELTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKP GQAPVLVIYRDSNRPSGIPERFSGSNSGNTATLTISRAQ AGDEADYYCQVWDSSTA 495 IGλV4-3 LPVLTQPPSASALLGASIKLTCTLSSEHSTYTIEWYQQR PGRSPQYIMKVKSDGSHSKGDGIPDRFMGSSSGADRYLT FSNLQSDDEAEYHCGESHTIDGQVG 496 IGλV4-60 QPVLTQSSSASASLGSSVKLTCTLSSGHSSYIIAWHQQQ PGKAPRYLMKLEGSGSYNKGSGVPDRFSGSSSGADRYLT ISNLQLEDEADYYCETWDSNT 497 IGλV5-39 QPVLTQPTSLSASPGASARFTCTLRSGINVGTYRIYWYQ QKPGSLPRYLLRYKSDSDKQQGSGVPSRFSGSKDASTNA GLLLISGLQSEDEADYYCAIWYSSTS 498 IGλV7-46 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYPYWFQ QKPGQAPRTLIYDTSNKHSWTPARFSGSLLGGKAALTLS GAQPEDEAEYYCLLSYSGAR 499 IGλV8-61 QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYQ QTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTIT GAQADDESDYYCVLYMGSGI 500 IGλV9-49 QPVLTQPPSASASLGASVTLTCTLSSGYSNYKVDWYQQR PGKGPRFVMRVGTGGIVGSKGDGIPDRFSVLGSGLNRYL TIKNIQEEDESDYHCGADHGSGSNFV 501 IGHD1-1 GGTACAACTGGAACGAC See (1) below. 502 IGHD1-14 GGTATAACCGGAACCAC 503 IGHD1-20 GGTATAACTGGAACGAC 504 IGHD1-7 GGTATAACTGGAACTAC 505 IGHD2-21_v1 AGCATATTGTGGTGGTGA T TGCTATTCC 506 IGHD2-21_v2 AGCATATTGTGGTGGTGA C TGCTATTCC Common allelic variant encoding a different amino acid sequence, compared to v1, in 2 of 3 forward reading frames. 507 IGHD2-8 AGGATATTGTACTAATGGTGTATGCTATACC 508 IGHD3-16 GTATTATGATTACGTTTGGGGGAGTTATGCTTATACC 509 IGHD3-9 GTATTACGATATTTTGACTGGTTATTATAAC 510 IGHD4-23 TGACTACGGTGGTAACTCC 511 IGHD4-4/4-11 TGACTACAGTAACTAC 512 IGHD5-12 GTGGATATAGTGGCTACGATTAC 513 IGHD5-24 GTAGAGATGGCTACAATTAC 514 IGHD6-25 GGGTATAGCAGCGGCTAC 515 IGHD6-6 GAGTATAGCAGCTCGTCC 516 IGHD7-27 CTAACTGGGGA (1) Each of the IGHD nucleotide sequences can be read in three (3) forward reading frames, and, possibly, in 3 reverse reading frames. For example, the nucleotide sequence given for IGHD1-1, depending on how it inserts in full V-DJ rearrangement, may encode the full peptide sequences: GTTGT (SEQ ID NO: 517), VQLER (SEQ ID NO: 518)and YNWND (SEQ ID NO: 519) in the forward direction, and VVPVV (SEQ ID NO: 520), SFQLY (SEQ ID NO: 521) and RSSCT (SEQ ID NO: 522) in the reverse direction. Each of these sequences, in turn, could generate progressively deleted segments as explained in the Examples to produce suitable components for libraries of the invention.

Example 15 Selection of Antibodies from the Library

In this example, the selection of antibodies from a library of the invention (described in Examples 9-11 and other Examples) is demonstrated. These selections demonstrate that the libraries of the invention encode antibody proteins capable of binding to antigens. In one selection, antibodies specific for “Antigen X”, a protein antigen, were isolated from the library using the methods described herein. FIG. 24 shows binding curves for six clones specifically binding Antigen X, and their Kd values. This selection was performed using yeast with the heavy chain on a plasmid vector and the kappa light chain library integrated into the genome of the yeast.

In a separate selection, antibodies specific for a model antigen, hen egg white lysozyme (HEL) were isolated. FIG. 25 shows the binding curves for 10 clones specifically binding HEL; each gave a Kd>500 nM. This selection was performed using yeast with the heavy chain on a plasmid vector and the kappa light chain library on a plasmid vector. The sequences of the heavy and light chains were determined for clones isolated from the library and it was demonstrated that multiple clones were present. A portion of the FRM3s (underlined) and the entire CDRH3s from four clones are shown below (Table 53 and Table 54, the latter using the numbering system of the invention).

TABLE 53 Sequences of CDRH3, and a Portion of FRM3, from Four HEL Binders SEQ Seq ID Name NO: FRM3 and CDRH3 Tail N1 DH N2 H3-JH CR080362 523 AKGPSVPAARAEYFQH G PS VPA AR AEYFQH CR080363 524 AREGGLGYYYREWYFDL E GGL GYY RE WYFDL Y CR080372 525 AKPDYGAEYFQH — P DYG — AEYFQH EK080902 526 AKEIVVPSAEYFQH E — IVV PS AEYFQH

TABLE 54 Sequences of CDRH3 from Four HEL Binders in Numbering System of the Invention, According to the Numbering System of the Invention [Tail] [N1] [DH] [N2] [H3-JH] Clones 95 96 96A 96B 97 97A 97B 97C 97D 98 98A 98B 99E 99D 99C 99B 99A 99 100 101 102 CDRH3 Length CR080362 G P S — V P A — — A R — — — — A E Y F Q H 14 CR080363 E G G L G Y Y Y — R E — — — — — W Y F D L 15 CR080372 — P — — D Y G — — — — — — — — A E Y F Q H 10 EK080902 E — — — I V V — — P S — — — — A E Y F Q H 12 Sequence Identifiers: CR080362 (SEQ ID NO: 523); CR080363 (SEQ ID NO: 524); CR080372 (SEQ ID NO: 525); EK080902 (SEQ ID NO: 526) The heavy chain chassis isolated were VH3-23.0 (for EK080902 and CR080363), VH3-23.6 (for CR080362), and VH3-23.4 (for CR080372). These variants are defined in Table 8 of Example 2. Each of the four heavy chain CDRH3 sequences matched a designed sequence from the exemplified library. The CDRL3 sequence of one of the clones (ED080902) was also determined, and is shown below, with the surrounding FRM regions underlined:

CDRL3: YYCQESFHIPYTFGGG. (SEQ ID NO: 527) In this case, the CDRL3 matched the design of a degenerate VK1-39 oligonucleotide sequence in row 49 of Table 33. The relevant portion of this table is reproduced below, with the amino acids occupying each position of the isolated CDRL3 bolded and underlined:

CDR Junction Degenerate SEQ Chassis Length type Oligonucleotide ID 89 90 91 92 93 94 95 96 97 VK1-39 9 1 CWGSAAWCATHC 307 LQ EQ ST FSY HNPRST IST P FY T MVTABTCCTTWC ACT

Example 16 Libraries Utilizing Non-Human DH Segments

This example illustrates a non-limiting selection of non-human vertebrate DH segments for use in the libraries of the invention. Non-human vertebrate DH segments were generally selected as follows. First, an exemplary survey of published IGHD sequences was performed as summarized below. Second, the degree of deletion on either end of the IGHD gene segments was estimated by analogy with human sequences (see Example 4.1). For the presently exemplified library, progressively deleted DH segments as short as three amino acids were included. As enumerated in the Detailed Description, other embodiments of the invention comprise libraries with DH segments with a minimum length of about 1, 2, 4, 5, 6, 7, 8, 9, or 10 amino acids.

Table 55 lists IGHD segments for a variety of species, namely Mus musculus (mouse; BALB/C and C57BL/6), Macaca mulatta (rhesus monkey), Oryctolagus cuniculus (rabbit), Rattus norvegicus (rat), Ictalurus punctatus (catfish), Gadus morhua L (Atlantic cod), Pan troglodytes (chimpanzee), Camelidae sp. (camel), and Bos sp. (cow). The sequences were obtained from the publications cited in Table 55. The DNA sequences encoding the IGHD genes are presented together with their translations in all three forward reading frames and, in some cases, three reverse reading frames. It will be appreciated that a skilled artisan could readily translate the reverse reading frames in those cases where they are not provided herein. Without being bound by theory, it is generally believed that the forward reading frames tend to be favored for inclusion in actual complete antibody sequences.

For the rat sequences, a procedure was implemented to extract the IGHD information from the most recent genomic assembly. First, the genomic location of a typical IGHV gene, e.g., 138565773 on chromosome 6, was identified from the literature (Das et al., Immunogenetics, 2008, 60: 47, incorporated by reference in its entirety). This location (i.e., 138565773 on chromosome 6) was then used to identify the contig and location within Genbank, and the approximately 150K bp upstream (because the genes of interest are in the minus strand) segment was extracted. Searches for canonical (e.g., mouse and human) recombination signal sequences (RSS) were conducted and candidate coding regions of lengths between about 10 and about 50 nucleotides were considered putative IGHD genes. The results of this IGHD gene identification process were consistent with the data that was available in the literature (e.g., the IGHD sequence designated “D15” in Table 55 is identical to the sequence highlighted in FIG. 3A of Brüggemann et al., Proc. Natl. Acad. Sci. USA, 1986, 83: 6075, incorporated by reference in its entirety). Finally, when the translation led to a stop codon, the longest open reading frame (ORF) was chosen to represent the peptide contribution. For example, translation in the first reverse reading frame (R1) of the rabbit sequence D2a results in the sequence *HKHNQHNHKYSN, where ‘*’ represents a stop codon; in such case the longest ORF would be HKHNQHNHKYSN, as reported in Table 55. Alternatively, in the case of long segments, such as those derived from the cow (see Table 55), appropriate sub-segments not comprising a stop codon would be considered. For example, translation of the cow DH1 gene in the first reading frame, provides MIR[stop]VWL[stop]LL[stop]CCY, which naturally would give rise to the ORFs or sub-segments: MIR, VWL, and CCY, when keeping a minimum length of three amino acids. The procedure used above for the rat, was also used for the chimpanzee (Pan troglodytes) and the three sets of sequences that were determined using the foregoing method are listed in Table 55. Only the forward reading frame translations are presented, but it will be appreciated that one of ordinary skill in the art could readily generate the corresponding reverse translations.

For each of the sequences set forth in the tables described above, variants may be generated by systematic deletion from the N- and/or C-termini, until there are three amino acids remaining. For example, for gene D6s 4 from the rhesus macaque, the full sequence GYSGTWN may be used to generate the progressive deletion variants: GYSGTW, GYSGT, GYSG, GYS, YSGTWN, SGTWN, GTWN, TWN, YSGTW, YSGT, YSG, SGTW, GTW, and so forth. This progressive deletion procedure is taught in detail herein in other parts of the specification. In general, and as shown in Example 4.1, for any full-length sequence of size N, there will be a total of (N−1)*(N−2)/2 variants, including the original full-length sequence, when the termini are progressively deleted to obtain a minimum of three amino acids per segment. The number of variants will increase or decrease accordingly, depending on the minimum length of the progressively deleted DH segment; e.g., (N−2)*(N−3)/2 for a minimum length of four and (N)*(N−1)/2 for a minimum length of two. This relationship can be generalized to (N+1-L)*(N+2-L)/2 where L is the number of amino acid residues in the shortest segment and L is always smaller than N. In the extreme case where L equals N, as expected, one obtains (1)*(2)/2, or just one segment, namely the original segment.

For the disulfide-loop-encoding segments, as exemplified by sequence D2S3 of rhesus translated in the second forward reading frame (AHCSDSGCSS), the progressive deletions were limited, in the present exemplification of the library, so as to leave the loop intact; i.e., only amino acids N-terminal to the first Cys, or C-terminal to the second Cys were deleted in the respective D segment variants; i.e., AHCSDSGCS, AHCSDSGC, HCSDSGCSS, CSDSGCSS, HCSDSGCS, HCSDSGC, CSDSGCS, and CSDSGC. This choice was made to avoid the presence of unpaired cysteine residues in the currently exemplified version of the library. For the same reason, segments with an odd number of Cys residues may be avoided in library construction. For example, the peptide segment resulting from the first reverse translation of the mouse (C57BL/6 strain) DST4 gene is SLSC, with the last Cys being potentially unpaired. This segment may be ignored, or considered only in its C-terminal deleted derivative, SLS. However, as discussed in the Detailed Description, other embodiments of the library may include unpaired cysteine residues, or the substitution of these cysteine residues with other amino acids.

According to the criteria outlined above and throughout the specification, a number of sequences, or subsets thereof, may be chosen for inclusion in a library of the invention. Selection of these segments may be carried out using a variety of criteria, individually or in combination. Exemplary non-limiting criteria include:

-   -   (a) choosing segments that are most diverse in length and         sequence;     -   (b) choosing segments with maximal “human string content” (see,         e.g., US Pub. No. 2006/0008883, incorporated by reference in its         entirety); or     -   (c) choosing segments with a minimal number of predicted T-cell         epitopes (see, e.g., U.S. Pat. No. 5,712,120, WO 9852976, and US         Pub. No. 2008/0206239, each of which is incorporated by         reference in its entirety).

TABLE 55 IGHD segments from other vertebrates Refer- Species Name DNA ence F1 F2 F3 R1 R2 R3 Mouse_C57BL/6 DFL16.1 TTTATTACTA [1] FITTVVAT LLLR YYYGSSY GSYYRSNK LLP VATTVVI CGGTAGTAG CTACC Mouse_C57BL/6 DSP2.2 TCTACTATG [1] STMITT LR YYDYD RNHSR S VVIIV ATTACGAC Mouse_C57BL/6 DSP2.3 TCTACTATG [1] STMVTT LLWLR YYGYD RNHSR P VVTIV GTTACGAC Mouse_C57BL/6 DSP2.5 TCTACTATG [1] STMVMT LLW YYGND HYHSR SLP VITIV GTAATGAC Mouse_C57BL/6 DSP2.9 TCTACTATG [1] STMVMT LLW YYGND HYHSR SLP VITIV GTAATGAC Mouse_C57BL/6 DSP2.X CCTACTATA [1] PTIVTT LL YYSNY SYYSR LL VVTIVG GTAACTAC Mouse_C57BL/6 DST4 ACAGCTCAG [1] TAQAT QLRL SSGY SLSC PEL VA GCTAC Mouse_C57BL/6 DST4.2 CACAGCTCG [1] HSSGY TARA QLGL VARAV SPSC PEL GGCTAC Mouse_C57BL/6 DQ52 CTAACTGGG [1] LTGT LG NWD PS SQL VPV AC Mouse_C57BL/6 P3 GAATACCTA [1] EYLP NTY IPT VF VGI GRYS CC Mouse_C57BL/6 P5 GACTACCTA [1] DYLP TTY LPT VV VGS GR CC Mouse_C57BL/6 P1 GAGTACCTA [1] EYLP STY VPT VL VGT GRYS CC Mouse_BALB/C DSP2.9 TCTATGATG [2] SMMVTT WLL YDGYY SNHHR PS VVTII GTTACTAC Mouse_BALB/C DSP2.2 TCTACTATG [3] STMITT LR YYDYD RNHSR S VVIIV ATTACGAC Mouse_BALB/C DSP2.5 TCTACTATG [3] STMVTT LLW YYGNY SYHSR LP VVTIV GTAACTAC Mouse_BALB/C DSP2.6 CCTACTATG [3] PTMVTT LLWLR YYGYD RNHSR S VVTIVG GTTACGAC Mouse_BALB/C DFL16.1 TTTATTACTA [3] FITTVVAT LLLR YYYGSSY SYYRSNK LLP VATTVVI CGGTAGTAG CTAC Mouse_BALB/C DSP2.3 TCTACTATG [3] STMVTT LLWLR YYGYD RNHSR P VVTIV GTTACGAC Mouse_BALB/C DFL16.2 TTCATTACTA [3] FITTAT SLLRL HYYGY SRSNE P VAVVM CGGCTAC Mouse_BALB/C DSP2.4 TCTACTATG [3] STMVTT LLWLR YYGYD RNHSR S VVTIV GTTACGAC Mouse_BALB/C DSP2.7 CCTACTATG [3] PTMVTT LLW YYGNY SYHSR LP VVTIVG GTAACTAC Mouse_BALB/C DSP2.8 CCTAGTATG [3] PSMVTT LVW YGNY SYHTR LPY VVTILG GTAACTAC Mouse_BALB/C DQ52 CTAACTGGGA [4] LTG LG NW PS SQL PV Mouse_BALB/C DST4 AGACAGCTC [5] RQLGL DSSG TARA PELS ARAV SPSCL GGGCTA Mouse_BALB/C DSP2.1 TCTACTATG [6] STMVTT LLW YYGNY SYHSR LP VVTIV GTAACTAC Mouse_BALB/C DSP2.x CCTACTATA [6] PTIVTT LL YYSNY SYYSR LL VVTIVG GGTAACTAC Rhesus D6S4 GGGTATAGC [7] GYSGTWN GIAARG RHVE VPRAAIP STCRYT FHVPLYP GGCACGTG GAAC Rhesus D6S3 GGGGTATAG [7] RWLV GYSGGWS GIAVAG DQPPLYP GPATAIP TSHRYTP CGGTGGCT GGTCC Rhesus D6S2 GGGTATAGC [7] GYSSWS GIAAG QLV GPAAIP TSCYT DQLLYP AGCTGGTCC Rhesus D6S1 GGGTATAGC [7] GYSSGWY GIAAAG QRLV VPAAAIP TSRCYT YQPLLYP AGCGGCTG GTAC Rhesus D5S5 GGGGATACA [7] GDTVGTVT GIQWVQL GYSGYSY NCTHCIP LYPLYP VTVPTVSP GTGGGTACA GTTAC Rhesus D5S4 GTGGTATAG [7] TTVT WYRLRL GIDYGY NRSLYH SIP VTVVYTT ACTACGGTT AC Rhesus D5S3 GGGGATATA [7] GDIVGTVT WVQL GYSGYSY NCTHYIP LYPLYP VTVPTISP GTGGGTACA GTTAC Rhesus D5S2 GTGGATACA [7] VDTATVT WIQLQL GYSYSY NCSCIH LYP VTVAVST GCTACAGTT AC Rhesus D5S1 GTGGATACA [7] VDTVGTVT WIQWVQL GYSGYSY NCTHCIH LYPLYP VTVPTVST GTGGGTACA GTTAC Rhesus D4S5 TGACTACGG [7] LR DYGNY TTVT LP VVTVV SYRS TAACTAC Rhesus D4S4 TGACTACGG [7] LRNL DYGI TTES P LDSVV RFRS AATCTAG Rhesus D4S3 TGACTACGG [7] QL DYGSSY TTVAA LLP VAATVV SCYRS TAGCAGCTA C Rhesus D4S2 TGAATACAG [7] IQ EYSNY NTVT LLYS VVTVF SYCI TAACTAC Rhesus D4S1 TGACTACGG [7] LR DYGNY TTVT LP VVTVV SYRS TAACTAC Rhesus D3S5 GTATTACTA [7] VLL YYYYSGSC ITIVVVVT QLPL VVTTTTIVI SNNYHYSNT TAGTGGTAG YY TTGTTACTA C Rhesus D3S4 GTATTACGA [7] VLRLRY YYDYDISS ITITILVV YRLLIS GLISTTNIVIVI YRNRNT TTACGATAT RY DIK TAGTAGTCG ATATTAAAC C Rhesus D3S3 GTATTACTA [7] LLL YYYSGSYY ITIVVVIT LPL VVITTTIVI SNNYHYSNT TAGTGGTAG Y TTATTACTAC Rhesus D3S2 GTATTACGA [7] LRLLLH YYEDDYGY ITRMITVT SSS GVIVTVIILVI CNSNRNHPRNT GGATGATTA YYT IT CGGTTACTA TTACACC Rhesus D3S1 GTATTACAA [7] VLQFLEWL YYNFWSGY ITIFGVVI PLQKL GVITTPKIVI CNNHSKNCNT TTTTTGGAG LH YT T TGGTTATTA CACC Rhesus D2S5 AGGATATTG [7] RILYCYYL GYCTATTC DIVLLLLV ARQVVAVQ TSSSSTIS QYNIL TACTGCTAC SS LA YP TACTTGTCT AGCC Rhesus D2S4 AGGATATTG [7] WWCLLH GYCSGGVC DIVVVVSA VEQTPPLQ GGADTTTTIS WSRHHHYNIL TAGTGGTGG ST P YP TGTCTGCTC CACC Rhesus D2S3 AGCACACTG [7] WLLL AHCSDSGC HTVVIVAA EEQPLSLQ GGAATITTVC RSSHYHYSVL TAGTGATAG SS P CA TGGCTGCTC CTCC Rhesus D2S2 AGCATATTG [7] SILLWWCL AYCCGGVC HIVVVVSA QTPPQQYA GVADTTTTIC CSRHHHNNML TTGTGGTGG LH YT T TGTCTGCTA CACC Rhesus D2S1 AGGATATTG [7] WWCLLR GYCSGGVC DIVVVVSA QTPPLQYP GVADTTTTIS RSRHHHYNIL TAGTGGTGG YA T TGTCTGCTA CGCC Rhesus D1S6 GGTATAACT [7] GITGTT LEL YNWNY SSSYT FQLY VVPVIP GGAACTAC Rhesus D1S5 GGTATAGCT [7] GIAGTT LER YSWND RSSYT SFQLY VVPAIP GGAACGAC Rhesus D1S4 GGTACAGCT [7] GTAGT VQLEL YSWNY SSSCT FQLY IVPAVP GGAACTAT Rhesus D1S3 GGTATAACT [7] GITGTT LER YNWND RSSYT SFQLY VVPVIP GGAACGAC Rhesus D1S2 GGAACACCT [7] GTPGTT EHLER NTWND GRSRCS SFQVF VVPGVP GGAACGACC Rhesus D1S1 GATATAGCT [7] DIAGTT LEQ YSWNN CSSYI LFQLY VVPAIS GGAACAAC Rabbit D1 TAGCTACGA [8] LR SYDDYGDY ATMTMVI SP VITIVIVA NHHSHRS TGACTATGG TGATTAC Rabbit D2a GTTACTATA [8] VTILMVML LLYLWLCW YYTYGYAG HKHNQHNH PA GSISITSITISIVT CTTATGGTT VMLML LCLCY YAYAT KYSN ATGCTGGTT ATGCTTATG CTACC Rabbit D2b GTTATGCTG [8] VMLVMLVM LCWLCWLW YAGYAGYG HNHNQHNQ VA GSITITSITSIT GTTATGCTG VMLP LCY YAT HN GTTATGGTT ATGCTACC Rabbit D3 GCATATGCT [8] AYASSSGY HMLVVVVI WLLY YIITTTTS VYNNHYY PLLLAYA AGTAGTAGT YI IY IC GGTTATTAT ATAC Rabbit D4 GTTACTATA [8] VTIVVAGV WLG YYSSGWG HPSHYYSN PQPLL TPATTIVT GTAGTGGCT GGGGTG Rabbit D5 GTTATGCTG [8] VMLVVVII LLY YAGSSYYT YNNYYQHN LLPA GIITTTSIT GTAGTAGTT ATTATACC Rabbit D6 GTTATGCTG [8] VMLVVAGM LCW YAGSSWD HPSYYQHN SQLLPA IPATTSIT GTAGTAGCT GGGATG Rabbit D7 ACTATGGTG [8] TMVI LW YGDY NHHS SP VITIV ATTAC Rat D1 TAAACTACA [9] TTIC KLQSA NYNLP ADCSL GRL WQIVV ATCTGCCA Rat D2 GGTATAATT [9] GIIRGT FGV YNSGY TPNYT YPELY VPRIIP CGGGGTAC Rat D3 GGTATAATT [9] GIIRG FGV YNSG TPNYT YPELY LPRIIP CGGGGTAA Rat D4 TTATAGATTA [9] INPK YRLILK ID FRINL SI LGLIY ATCCTAAAG Rat D5 TACATACTA [9] YILWV TYYGYNY HTMGIT LYP VVIPIVC SYTHSM TGGGTATAA CTAC Rat D6 TTTATAACAA [9] FITTT QL YNNY SCYK LL VVVI CTAC Rat D7 TCCTCAGGT [9] SSGESCVW PQVSPVSG VLCL PDTGLT PRHRTHLR QTQDSPE GAGTCCTGT GTCTGGG Rat D8 GGATATCTAG [9] GYL DI IS IS LDI RYP Rat D9 TTAACTACG [9] LTTEGIV LRRV NYGGYSE HYTLRS SLYPP LTIPSVV GAGGGTATA GTGAG Rat D10 TTTTTAACTA [9] FLTTVAT LQ FNYSSY SYCS LK VATVVK CAGTAGCTAC Rat D11 TTTATTACTA [9] FITMMVVI LLL YYYDGSYY SNNYHHSN LPS VVITTIIVI TGATGGTAG TT Y K TTATTACTAC Rat D12 GGATACCTAT [9] GYL DTY IP VS IGI RYP Rat D13 TTCATACTAT [9] FILWV SYYGYDY HTMGMT SYP VVIPIV SHTHSM GGGTATGAC TAC Rat D14 TTTATTACTA [9] FITMMVII WLLS YYYDGYYH DNNHHSNK PS VIITIIVI TGATGGTTA T TTATCAC Rat D15 CTAACTGGG [9] LTG LG NWE PS SQL LPV AG Rat D16 TTTATGTATA [9] FMYTTDYY LCILRIIT YVYYGLLL VVIIRSIH SNNP SVVYI CTACGGATT Y K ATTACTAC Catfish DH1 GTTATAGCA [10] VIAAGV QLG YSSWG YPSCYN LPQLL TPAAIT GCTGGGGTA G Catfish DH2 CAATATAGC [10] QYSG NIAG R PAIL TRYI PLY GGGT Catfish DH3 ATAACTACG [10] ITTA LR NYG RSY P AVV GC Catfish AF068137 TCGCGTGGC [11] SRGQ RVA AWP LATR GHA WPR CAA Atlantic cod core1 ATACAACTG [12] IQLGWG YNWAG TTGLG PAQLY PSPVV PQPSC GGCTGGGG Atlantic cod core2a ATACAGTGG [12] IQWGD YSGGI TVGG IPPLY DPPTV SPHC GGGGATC Atlantic cod core2b ATACAGTGG [12] IQWG YSG TVG TPLY PTV PHC GGT Atlantic cod core4 ATACAGGGG [12] IQGG YRG TGG PLY PPV PPC GG Atlantic cod core5a ATACGGGG [12] IRGD YGGI TGG IPPY DPPV SPR GGATC Chimpanzee Chimp_6224 CTAACTGGG [13] LTG LG NWG GA Chimpanzee Chimp_10468 TGACTACAG [13] LQ DYSNY TTVT TAACTAC Chimpanzee Chimp_10580 TGACTACGG [13] LR DYGDY TTVT TGACTAC Chimpanzee Chimp_30856 TGACTACGG [13] LR DYGDY TTVT TGACTAC Chimpanzee Chimp_173 GGTATAACT [13] GITGS LDR YNWID GGATCGAT Chimpanzee Chimp_474 GGTATAACT [13] GITGTT LEL YNWNY GGAACTAC Chimpanzee Chimp_1395 GAATATCTA [13] EYL NI IS Chimpanzee Chimp_1484 GAATACCCC [13] EYP NT IP Chimpanzee Chimp_5696 GGTATAACT [13] GITGTT LER YNWND GGAACGAC Chimpanzee Chimp_429 GGGTATAGC [13] GYSSGWY GIAVAG QWLV AGTGGCTGG TAC Chimpanzee Chimp_1045 GGGTATAGC [13] GYSGSWY GIAAAG RQLV GGCAGCTG GTAC Chimpanzee Chimp_4178 CCATGGGTG [13] PWV HGCSGY MGVVA TAGTGGCTA C Chimpanzee Chimp_8658 TGACTACGG [13] LR DYGNY TTVT TAACTAC Chimpanzee Chimp_11102 TGACTACGG [13] LR DYGNY TTVT TAACATAC Chimpanzee Chimp_2093 AGCATATTG [13] SILWW AYCGGDCY HIVVVTAM TGGTGGTGA A CTGCTATGC C Chimpanzee Chimp_4876 GGTGTAGTG [13] GVVAT WL CSGY GCTAC Chimpanzee Chimp_10664 GATATGGTG [13] DMVAT IWWL YGGY GCTAC Chimpanzee Chimp_11497 GCCTGAGAT [13] DPQDAA PEIPRTQH LRSPGRS CCCCAGGAC GCAGCAC Chimpanzee Chimp_25802 GGCGTGTGA [13] GV ACE RVR GAG Chimpanzee Chimp_5740 GTGGATATA [13] VDIVATIT WLRL GYSGYDY GTGGCTACG ATTAC Chimpanzee Chimp_7586 GTGGATATA [13] VDIVATIT WLRL GYSGYDY GTGGCTACG ATTAC Chimpanzee Chimp_9253 GTGGATATA [13] VDIVATIT WLRL GYSGYDY GTGGCTACG ATTAC Chimpanzee Chimp_9731 GTGGATACA [13] VDTATIT WIQLRL GYSYDY GCTACGATT AC Chimpanzee Chimp_14017 GTGGATATA [13] VDIVATIT WLRL GYSGYDY GTGGCTACG ATTAC Chimpanzee Chimp_84128 GTGGAGATG [13] VEMATIT WRWLQL GDGYNY GCTACAATT AC Chimpanzee Chimp_23293 GACCGCCAC [13] DRH TAT PP A Chimpanzee Chimp_1702 ATAGTGGTG [13] IVVVS WWC SGGV GTGTC Chimpanzee Chimp_638 AGAATAGCT [13] RIAGSKTL LGPKLSW NSWVQNSP GGGTCCAAA LA G ACTCTCCTG GC Chimpanzee Chimp_1760 AGAATAGCT [13] RIAGSKTL LGPKLSW NSWVQNSP GGGTCCAAA LA G ACTCTCCTG GC Chimpanzee Chimp_6453 ATCTTTTGA [13] KFALC SFESLPCA LLKVCPV AAGTTTGCC CTGTGCC Chimpanzee Chimp_8535 TTAGGATTT [13] LRPQ DFD RILIEAT TGATTGAGG CCACAG Chimpanzee Chimp_30042 GCAGGCTG [13] AGCGEGPG QAAGKDQG RLRGRTR CGGGGAAG GACCAGGG A Chimpanzee Chimp_44108 GTGGTGTC [13] VVS WC GV Camel camD4 ACTATAGCG [14] TIATM RL YSDY ACTATG Llama n/a CTAACTGGA [15] LTGA LEP NWS GCCA Cow DH1 ATGATACGA [16] MIR YDRCGCSY DTIGVVVV TAGGTGTGG [stop] CSVA IVVLL TTGTAGTTA VWL TTGTAGTGT [stop]LL TGCTAC [stop] CCY Cow DH2 GTAGTTGTC [16] VVVLMVIV LS[stop] SCPDGYSY CTGATGGTT MVMVVVMV WL[stop] GYGCGYGY ATAGTTATG MVVVVMIV LWLWLWLW GCSGYDCY GTTATGGTT MVMVVMVV LWL GYGGYGG GTGGTTATG MVVMVIVV [stop]WL YGGYGYSS GTTATGGTT IVIVILTN [stop]LL YSYSYTYE GTAGTGGTT I WLWWLWWL Y ATGATTGTT WWLWL ATGGTTATG [stop] GTGGTTATG [stop]L GTGGTTATG [stop]L GTGGTTATG [stop]LY GTTATAGTA LRI GTTATAGTT ATAGTTATA CTTACGAAT ATA Cow DH3 GTAGTTGTT [16] VVVIVVMV LL[stop] SCYSGYGY ATAGTGGTT MVVVMVMV WLWLWLWL GCGYGYGY ATGGTTATG MII WLWL DY GTTGTGGTT [stop]LY ATGGTTATG GTTATGATT ATAC Each of the following references are incorporated by reference in their entirety: [1] Ye, Immunogenetics, 2004, 56: 399; [2] Shimiza and Yamagishi, EMBO J, 1992, 11: 4869; [3] Kurosawa et al., Nature, 1981, 290: 565; [4] Dirkes et al., Immunogenetics, 1994, 40: 379; [5] Gerondakis et al. Immunogenetics, 1988, 28: 255; [6] Gu et al., Cell, 1991, 65: 47; [7] Link et al., Immunogenetics, 2002, 54: 240; [8] Friedman et al., J. Immunol., 1994, 152: 632; [9] GI code: 62651567; reverse strand 33906161-33793435; [10] Hayman et al., J. Immunol., 2000, 164: 1916; [11] Ghaffari and Lobb, J. Immunol. 1999, 162: 1519; [12] Solem and Stenvik, Dev. Comp. Immunol., 2006, 30: 57; [13] GI code: 114655167; reverse strand 203704-97555; [14] Nguyen et al., EMBO J, 2000, 19: 921; [15] GI code: 13345163; [16] Shojaei et al., Mol. Immunol., 2003, 40: 61.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

APPENDIX A GI Numbers of Kappa Light Chains Used to Derive the VK Libraries 23868 2385488 16923194 58222611 70798854 98956311 32779 2385490 16923202 58222613 70798856 98956323 32810 2385492 16923208 58222615 70798858 98956325 33059 2385494 17226623 58222617 70798860 98956327 33144 2385495 17226631 58222619 70798862 98956337 33156 2385497 17226635 58222621 70798866 98956341 33170 2597932 17226639 58222623 70798868 98956343 33173 2597935 17226643 58222625 70798872 98956349 33183 2597937 17226645 58222627 70798874 98956355 33185 2597943 17226655 58222629 70798878 98956357 33189 2597946 17381491 58222631 70798880 98956365 33191 2597948 17385013 58222633 70798882 98956375 33195 2597950 17385015 58222635 70798884 98956379 33200 2597952 17385017 58222637 70798886 98956381 33202 2599531 17385019 58222639 70798888 98956383 33221 2599533 17385021 58222641 70798890 98956400 33227 2599535 17483729 58222643 70798892 98956404 33230 2599545 18025561 58222645 70798894 98956406 33233 2625059 18025563 58222647 70798896 98956414 33237 2632152 18025573 58222649 70798898 98956418 33268 2654047 18025575 58222651 70798900 98956422 33288 2654051 18025577 58222653 70798902 98956426 33290 2654055 18025579 58222655 70798904 98956428 33294 2773084 18025581 58222657 70798906 98956430 33296 2920359 18025583 58222659 70798914 98956432 33298 2995674 18025585 58222661 70798916 98956436 33300 2995676 18025587 58222663 70798918 98956440 33302 2995678 18025589 58222665 70798920 99022977 33304 2995680 18025591 58222667 70798922 99022979 33324 2995682 18025593 58222669 70798926 99022981 33330 2995688 18025595 58222671 70798928 99022983 33415 2995690 18025597 58222673 70798930 99022985 33416 3023134 18025599 58222675 70798934 99022987 33417 3023136 18025603 58222677 70798936 99022989 33418 3023138 18025605 58222679 70798940 99022991 33421 3023140 18025607 58222681 70798942 99022993 33422 3023142 18025611 58222683 70798946 99022995 33423 3023144 18025613 58222685 70798948 99022997 33424 3023146 18025617 58222687 70798950 99022999 33426 3023148 18025621 58222689 70798952 99023002 33647 3251385 18025623 58222691 70798954 99023004 33649 3251387 18025627 58222693 70798956 99023006 33655 3251389 18025629 58222695 71058688 99023008 33657 3251391 18025635 58222697 71058704 99023010 33659 3251744 18025639 58222699 71058712 99023012 33665 3251749 18025641 58222701 71058717 99023474 33669 3251983 18025645 58222703 71058719 99023476 33679 3251985 18025651 58222705 71058721 99023478 33683 3288824 18025653 58222707 71058723 99023480 33685 3378165 18025655 58222709 71058725 99023482 33756 3378177 18025657 58222711 71058727 99023484 34022 3378183 18025659 58222713 71058729 99025082 36657 3451194 18025661 58222715 71058731 99025083 37860 3603382 18025665 58222717 71482591 99025084 37909 3603384 18025667 58222719 71482622 99025903 38361 3603386 18025669 58222721 71482624 99025916 38362 3603388 18025677 58222723 71482634 99026398 38363 3603390 18025679 58222725 71482636 99026399 38367 3603392 18025681 58222727 71482638 99026416 38436 3603394 18025683 58222729 71482640 99026418 38438 3603396 18025685 58222731 71482642 109240611 38439 3641303 18025687 58222733 71482644 109240615 38440 3641307 18025689 58222735 71482646 109240619 38441 3644015 18025693 58222737 71482648 109240627 38442 3644021 18025697 58222739 71482650 109240631 38448 3746530 18025701 58222741 71482652 109240635 38485 3747011 18025705 58222743 71482654 109240637 38487 3747015 18025709 58222745 71792302 109240641 38489 3821085 18025715 58222747 71792306 109240643 38491 3821088 18025717 58222749 71792308 109240647 38493 3901025 18025719 58222751 73532341 109240655 38495 3928173 18092607 58222753 75707120 109240657 38497 3928181 18092609 58222755 75707124 109240661 38499 3928185 18092611 58222757 75707126 109240665 38501 3928189 18092613 58222759 75707128 109240669 38503 3928210 18092615 58222761 75707130 109240671 38505 3928211 18092617 58222763 75707132 109240675 178678 3928212 18092619 58222765 75707134 109240679 182338 3928214 18092621 58222767 75707138 109240687 182340 3928215 18092623 58222769 75707140 109240691 182342 3928219 18307263 58222771 75707148 109240695 182344 3928220 18307265 58222773 75707154 109240701 182346 3928222 18307267 58222775 75707156 109240705 182348 3928223 18307269 58222777 75707158 109240709 183962 3928224 18307271 58222779 75707160 109240713 183968 3928225 18307273 58222781 75707162 109240717 183972 3928227 18307275 58222783 75707168 109240721 185375 3928231 18307277 58222785 75707170 109240723 185377 3928232 18307279 58222787 75707172 109240729 185379 3928233 18307281 58222789 75707174 109240733 185381 3928234 18307283 58222791 75707176 109240737 185383 3928235 18307285 58222793 75707180 109240741 185385 3928236 18307289 58222795 75707188 109240745 185387 3928237 18307291 58222797 75707194 109240760 185389 3928238 18307293 58222799 75707196 109240764 185391 3928239 18626727 58222801 75707198 109240766 185393 3928240 18626728 58222803 75707204 109240770 185395 3928243 18626729 58222805 75707206 109241210 185397 3928244 18626730 58222807 75707208 109241212 185399 3928245 18632678 58222809 75707210 109241214 185401 3928248 18698406 58222811 75707220 109241216 185403 3928250 19170347 58222813 75707222 109241218 185415 3928251 19701578 58222815 75707226 109241220 185417 3928252 19744467 58222817 75707228 109241450 185419 3928253 19744471 58222819 75707230 109241549 185423 3928254 19744475 58222821 75707232 109241551 185427 3928257 19744479 58222823 75707234 109242373 185811 3928258 19744487 58222825 75707236 109242377 185813 3928259 19744491 58222828 75707238 109242379 185815 3928260 19744495 58222830 75707240 109242381 185816 3928261 19744499 58222832 75707242 109242383 185827 3928263 19744503 58222834 75707244 109242385 185829 3928264 19744507 58222836 75707246 109242387 185831 3928265 19744511 58222838 75707248 109242389 185833 3928266 19744515 58222840 75707250 109242395 185835 3928267 19744519 58222843 75707262 109242399 185837 3928276 19744523 58222845 75707264 109242401 185839 3928277 19744527 58222847 75707268 109242403 185841 3928278 19744531 58222849 75707270 109242409 185845 3928279 19744535 58222851 75707272 109242411 185847 3928280 19744539 58222853 75707274 109242417 185849 3928283 19744543 58222855 75707276 109242419 185855 3928287 19744547 58222857 75707278 109242421 185859 3928288 19744551 58222859 75707282 109242423 185862 3928289 19744555 58222861 75707284 109242425 185866 3928290 19744559 58222863 75707292 109242427 185868 3928291 19744563 58222865 75707298 109245190 185870 3928293 19744567 58222867 75707300 109245192 185872 3928294 19744571 58222869 75707302 109245194 185874 3928295 19744575 58222871 75707304 109693080 185880 3928296 19744579 58222873 75707306 109693082 185882 3928297 19744583 58222875 75707316 109693084 185884 3928298 19744587 58222877 75707318 109693094 185886 3928299 20372497 58222879 75707322 109693096 185888 3928301 20372499 58222881 75707324 109693100 185890 3928302 20372501 58222883 75707334 109693102 185892 3928303 20372503 58222885 75707338 109693110 185894 3928304 20372505 58222887 75707340 109693112 185896 3928308 20372507 58222889 75707362 109693114 185898 3928309 20372509 58222891 75707368 109693116 185904 3928310 20372511 58222893 75707370 109693118 185906 3928312 20372513 58222895 75707372 109693120 185908 3928315 20372515 58222897 75707374 109693135 185910 3928316 20372517 58222899 75707378 109693137 185912 3928317 20372519 58222901 75707382 109693139 185920 3928318 20372521 58222903 75707384 109693144 185922 3928319 20372523 58222905 75707386 109693146 185928 3928320 20372525 58222907 75707398 109693148 185934 3928321 20372527 58222909 75707406 109693150 185950 3928323 20372529 58222911 75707408 109693152 185980 3928324 20387057 58222913 75707410 109693154 185984 3928325 20387059 58222915 75707412 109693157 185987 3928326 20387061 58222917 75707416 109693159 185988 3928327 21311286 58222919 75707418 109693165 186008 3928329 21311288 58222923 75707420 109693167 186015 3928330 21311294 58222925 75707422 109693169 186017 3928331 21311296 58222927 75707424 109693171 186019 3928332 21311318 58222929 75707426 109693177 186040 3928333 21311322 58222931 75707428 109693179 186041 3928334 21669062 58222933 75707430 109693181 186042 3928335 21669064 58222935 75707432 109693183 186047 3928336 21669066 58222937 75707434 109693187 186199 3928337 21669068 58222939 75707444 109693189 186266 3928338 21669070 58222941 75707446 109693201 254719 3928339 21669072 58222943 75707448 109693203 257550 3928340 21669074 58222945 75707454 109693206 261239 3928341 21669076 58222947 75707460 109693210 265236 3928342 21669078 58222949 75707462 109693216 265240 3928343 21669080 58222951 75707464 109693218 298552 3928344 21669082 58222953 75707472 109693220 298560 3928345 21669084 58222955 75707476 109693222 298827 3928346 21669086 58222957 75707500 109693228 298829 3928347 21669088 58222959 75707502 109693230 299955 3928348 21669090 58222961 75707504 109693232 306919 3928349 21669092 58222963 75707506 109693235 306957 3928350 21669094 58222965 75707508 109693237 306959 3928351 21669096 58222967 75707510 109693239 306961 3928352 21669098 58222969 75707514 109693241 306963 3928353 21669100 58222971 75707516 109693249 306965 3928354 21669102 58222973 75707518 109693253 306967 3928355 21669104 58222975 75707520 109693255 306971 3928356 21669106 58222977 75707522 109693261 306980 3928357 21669108 58222979 75707524 109693264 306982 3928358 21669110 58222981 75707526 109942421 306984 3928359 21669112 58222983 75707528 109942431 306986 3928360 21669114 58222985 75707530 110290934 306988 3928361 21669116 58222987 75707534 110610132 306990 3928362 21669118 58222989 75707536 110624509 306992 3928363 21669120 58222991 75707540 110657101 306994 3928364 21669122 58222993 75707542 110657103 306996 3928365 21669124 58222995 75707544 110657105 306998 3928366 21669126 58222997 75707546 110657107 307000 3928367 21669128 58222999 75707548 110657109 348203 3928368 21669130 58223001 75707550 110657111 348205 3928369 21669132 58223003 75707552 110657113 348207 3928370 21669134 58223005 75707586 110657115 348211 3928371 21669136 58223007 75707598 110657123 386052 3928372 21669138 58223009 75707600 110657124 396631 3928373 21669140 58223011 75707602 110657125 397787 3928374 21669142 58223013 75707604 110657158 397789 3928375 21669144 58223015 75707618 110657159 397791 3928376 21669146 58223017 76058957 110657160 397793 3928377 21669148 58223019 76252624 110657161 397795 3928378 21727250 58223021 76252626 110657162 398490 3928379 21998806 58223023 76252630 110657163 398491 3928380 21998808 58223025 76252632 110657164 398492 3928381 21998810 58223027 76252634 110657165 404110 3928382 21998812 58223029 76252636 110657166 404112 3928383 21998814 58223031 76252638 110657167 404114 3928384 21998816 58223033 76252640 110657168 408365 3928385 21998818 58223035 76252642 110657169 409042 3928386 21998820 58223037 76252644 110657170 414035 3928387 21998822 58223039 76252646 110657171 415651 3928388 21998824 58223041 76781673 110657172 415710 3928389 21998826 58223043 77378090 110657173 415955 3928390 21998830 58223045 77378092 110657174 415957 3928391 21998832 58223047 77378094 110657175 415959 3928392 22086572 58223049 77378096 110657176 415961 3928393 22086575 58223051 77378098 110657177 415963 3928394 22086581 58223053 77378100 110657178 415965 3928395 22086587 58223055 77378102 110657179 415967 3928396 22086593 58223057 77378105 110657180 415969 3928397 22091617 58223059 77378107 110657181 415971 3928398 22214019 58223061 77378109 110657182 416329 3928399 22214023 58223063 77378111 110657183 416331 3928400 22297542 58223065 77378135 110657184 416333 3928401 22556681 58223067 77378137 110657185 416335 3928402 22556683 58223069 77378139 110657186 416337 3928403 22556684 58223071 77378141 110657187 430845 3928404 22607990 58223073 77378143 110657188 431039 3928405 22620896 58223075 77378145 110657189 431040 3928406 22620899 58223077 77378147 110657230 431041 3928407 22640510 58223079 77378149 110657232 431042 3928408 22640512 58223081 77378151 110657234 431043 3928409 22640513 58223083 77378153 110657236 431044 3928410 22642789 58223085 77378155 110657238 431045 3928411 22642790 58223087 77378157 110657240 431046 3928412 22642791 58223089 77378159 110657242 431047 3928413 22642808 58223091 77378161 110657244 431048 3928414 22642809 58223093 77378163 110657246 431049 3928415 22642810 58223095 77378165 110657248 431051 3928416 22642811 58223097 77378167 110657250 431052 3928417 22643188 58223099 77378169 110657252 431053 3928418 22643190 58223101 77378172 110657254 431067 3928419 22643192 58223103 77378174 110657256 431069 3928420 22643196 58223105 77378176 110657258 431071 3928421 22647625 58223107 77378224 110657615 431073 3928422 22647633 58223109 77378225 110657617 431075 3928423 23194480 58223111 77378228 110657619 431077 3928424 23194500 58223113 77378230 110657621 431079 3928425 23225992 58223115 77378234 110657624 431081 3928426 23225994 58223117 77378236 110657676 431083 3928427 23225996 58223119 77378237 110657678 431085 3928428 23234613 58223121 77378239 110657728 431087 3928430 23320663 58223123 77378241 110657730 431089 3928431 23342423 58223125 77378245 110658341 433889 3928432 23343554 58223127 77378247 110660158 436562 3928433 24412754 58223129 77378249 110660166 440153 3928434 24412756 58223131 77378251 110660174 441312 3928435 24412758 58223133 77378253 112184495 441314 3928436 24474081 58223135 77378255 112184497 441316 3928437 24850297 58223137 77379405 112184499 441318 3928438 26985941 58223139 77379407 112184501 441320 3928439 27368974 58223141 77379409 112184503 441322 3928440 27368976 58223149 77379412 112184505 441324 3928441 27368978 58223151 77379414 112184507 441330 3928442 27368981 58223153 77379416 112184509 441332 3928443 27368983 58223155 77379418 112184511 441334 3928444 27368986 58223157 77379420 112184513 441336 4100379 27368991 58223159 77379422 112189154 441338 4100381 27368993 58223161 77379425 112191695 441342 4100383 27368997 58223163 77379427 112191699 441344 4103644 27368999 58223165 77379429 112703827 441346 4103662 27369001 58223167 77379431 112708249 441348 4103664 27369003 58223169 77379433 112708250 441350 4103666 27369007 58223171 77379435 112711584 441352 4103674 27369009 58223173 77379437 112712351 441354 4128063 27369011 58223175 77379439 112712352 441356 4139195 27818830 58223177 77379441 112712353 441358 4139197 27867541 58223179 77379443 112712354 441360 4139199 27873542 58223181 77379445 112712355 441364 4139201 27875080 58223183 77379447 112712356 441366 4323178 27875088 58223185 77379449 112712357 441368 4323182 27875191 58223187 77379457 112712358 441370 4323186 27875199 58223189 77379459 112712359 441372 4323194 28611056 58223191 77379461 112712360 441374 4323809 28848873 58223193 77379463 112712361 441376 4323811 28883544 58223195 77379477 112712362 441378 4323813 28883548 58223197 77379479 112712363 441380 4323821 28883550 58223199 77379481 112712364 441382 4323823 29650328 58223201 77379483 112712365 441384 4323825 29650334 58223203 77379485 112712366 441386 4323829 29650337 58223205 77379487 112712367 441388 4323831 29650339 58223207 77379489 112712368 441390 4323833 29725711 58223209 77379491 112712369 441392 4323839 29725713 58223211 77379493 112712370 441394 4323841 29725715 58223213 77379495 112712371 441396 4323845 29725717 58223215 77379497 112712372 441398 4323847 29725719 58223217 77379499 112712373 441400 4323849 29725721 58223219 77379501 112712374 441402 4323851 29725723 58223221 77379503 112712375 441408 4323853 29725725 58223223 77379505 112712376 441412 4323855 29725727 58223225 77379507 112712377 441414 4323857 29725729 58223227 77379509 112712378 441416 4323859 29725731 58223229 77379511 112712379 441418 4323861 29725733 58223231 77379513 112712380 441422 4323863 30026987 58223233 77379515 112712381 441424 4323865 30258344 58223235 77379517 112712382 441426 4323869 30258346 58223237 77379519 112712383 441428 4323871 30793253 58223239 77379521 112727205 441430 4323873 30793255 58223241 77379523 112727206 441432 4323875 30793257 58223243 77379525 112727207 441434 4323877 30793259 58223245 77379527 112727208 441436 4323881 30793261 58223247 77379529 112727209 441440 4323883 30793263 58223249 77379545 112727210 441444 4323885 30793265 58223251 77994607 112727211 441446 4323887 30793565 58223253 77994611 112727212 441448 4323889 30793567 58223255 77994615 112727213 452060 4323891 30793569 58223257 77994619 112727214 452061 4323893 30793571 58223259 78629976 112727215 452062 4323895 30793573 58223261 78629977 112727216 452063 4323897 30841928 58223263 78629978 112727217 459655 4323899 30841931 58223265 80750467 112727218 460858 4323901 30841933 58223267 80975580 114155738 472970 4323903 30841935 58223269 80975600 114155883 472971 4323905 30841939 58223271 80975604 114155884 472972 4323907 30841943 58223273 80975616 114156208 472973 4323909 30841945 58223275 80975618 114207907 472974 4323911 30841947 58223277 80975638 114385493 472975 4323913 31879463 58223279 80975642 114385505 472976 4323915 31879464 58223281 80975644 114385507 487826 4323923 31879467 58223283 81020146 114385509 487827 4323927 31879468 58223285 81020229 114385511 493148 4323929 31879471 58223287 81020258 114385513 493149 4323931 31879472 58223289 81239122 114385515 493150 4323933 33021483 58223291 81251581 114385517 496044 4323935 33044572 58223293 81251585 114385521 496046 4323937 33044573 58223295 82794837 114385537 496048 4323939 33044574 58223297 83410334 114385539 496050 4323941 33044582 58223299 83697271 114385541 496053 4323945 33044586 58223301 83959521 114385543 496055 4323947 33051527 58223303 83959523 114385545 496059 4323949 33051528 58223305 83959525 114385547 496061 4323951 33070272 58223307 83959937 114385549 496063 4323953 33070283 58223309 83959939 114385551 496065 4323955 33070284 58223311 83964685 114385553 496071 4323957 33083474 58223313 83964762 114385567 496073 4323959 33083476 58223315 83964764 114385569 506420 4323961 33083477 58223317 83964766 114385571 506424 4323963 33083478 58223319 83964768 114385573 510839 4323965 33083479 58223321 83966574 114385575 510841 4323983 33083480 58223323 83966576 114385579 510843 4323989 33083481 58223325 83966578 114385581 510845 4323993 33083482 58223327 83966655 114385583 514428 4323997 33083483 58760238 83966657 114385585 514429 4323999 33085842 59890568 83966659 114385587 514430 4324005 33235609 59890571 83966661 114385589 514431 4324007 33235611 59894819 83966663 114385591 514432 4324009 33235613 60392126 83966665 114385593 514433 4324011 33235615 60616327 83966667 114385595 514434 4324013 33235617 60616352 83970756 114385597 515780 4324019 33235619 60650119 83970763 114385599 516137 4378181 33235621 60650123 83970769 114385601 516187 4378183 33235623 60734312 83970772 114385603 516198 4378185 33235625 61697118 84659318 114385605 516213 4378187 33235627 61853816 84659320 114385607 516249 4378189 33235629 61970154 84660715 114385609 516265 4378191 33235631 61970158 84660717 114385611 516316 4378193 33235633 61970160 84660719 114385613 545722 4378195 33304656 61970164 84660720 114385615 557650 4378197 33304658 61970168 84660721 114385617 557651 4378199 33304661 61970172 84660722 114385619 560677 4378201 33304663 61970176 84660723 114385621 560678 4378203 33355480 61970180 84660725 114385623 560841 4378207 33868634 61970184 84797793 114385625 560843 4378209 33868636 61970192 84797795 114385627 575228 4378211 33868638 61970194 84797797 114385629 575236 4378213 33868640 61970198 84797799 114385631 575240 4378215 33868642 61970202 84797801 114385633 575257 4378217 33868644 61970206 84797803 114385635 575261 4378221 33868646 61970228 84797805 114385645 587143 4378223 37287525 62001845 84797807 114385647 587245 4378225 37605051 62120916 84797823 114385649 587323 4378227 37694620 62120917 84797825 114385651 587325 4378229 37694622 62120918 84797827 114385653 587327 4378233 37694624 62120919 84797857 114385655 587329 4378237 37694626 62120920 84797861 114385659 587331 4378239 37694628 62120921 84797883 114385661 587333 4378243 37694630 62120922 84797915 114385663 587335 4378245 37694632 62120923 84797929 114385665 587337 4378247 37694634 62120924 84797959 114385669 587341 4378249 37694636 62120925 84797961 114385671 587343 4378251 37694638 62120926 84797963 114385673 587345 4378253 37694640 62120927 84797979 114385675 587347 4378255 37694642 62120929 84797981 114385677 587349 4378259 37694644 62120931 84797985 114385679 587351 4378261 37694646 62120932 84798001 114385681 587353 4378265 37694648 62120933 84798003 114385683 598165 4378267 37694650 62120934 84798005 114385685 598167 4378269 37694654 62120935 84798007 114385687 598170 4378271 37694660 62120938 84798009 114385689 598172 4378273 37694662 62120939 84798011 114385691 601979 4378275 37694664 62120940 84798033 114385693 601982 4378279 37694666 62120941 84798035 114385699 601984 4378281 37694668 62120943 84798055 114385701 609002 4378283 37694670 62120944 84798057 114385703 609004 4378287 37694672 62120945 84798059 114385705 619259 4378291 37694674 62120946 84798061 114385707 623043 4378293 37694676 62120947 84798063 114385709 624874 4378295 37694678 62120948 84798103 114385711 632983 4378297 37694680 62120949 84798107 114385713 632985 4378299 37694682 62120950 84798115 114385715 632987 4378301 37694684 62120951 84798117 114385717 633227 4378303 37694686 62120952 84798147 114385719 642581 4378305 37694688 62120953 84798149 114385721 681896 4378307 37694690 62120954 84798167 114385723 681899 4378309 37694692 62120955 84798169 114385725 685029 4378313 37694694 62120956 84798171 114385727 693862 4378315 37694696 62120957 84798173 114385729 722413 4378317 37694698 62120958 84798175 114385731 722417 4378319 37694700 62120959 84798177 114385744 722419 4378323 37694702 62120960 84798179 114385746 722421 4378325 37694704 62120961 84798181 114385748 722423 4378327 37694706 62120962 84798183 114385750 722425 4378331 37694708 62199500 84798197 114385752 722427 4378333 37694710 62421462 84798199 114385756 722429 4378335 37702652 62421466 84798201 114385774 722431 4378337 37732215 62720427 84798203 114385776 722433 4378339 37780362 62720431 84798213 114385778 722435 4378341 39103877 62720436 84798215 114385780 722437 4378343 39103879 62720442 84798217 114385782 722439 4378345 39103881 62720444 84798219 114385804 722441 4378347 39103883 62720446 84798241 114385806 722443 4378349 39103885 62720452 84798249 114385808 722455 4378351 39103887 62720454 84798255 114385921 722461 4378353 40231616 62720473 84798257 115268711 722463 4378359 40288410 62720475 84798267 115268713 722465 4378361 40288412 62720477 84798269 115268880 722467 4378363 40288414 62720483 84798271 115268892 722469 4378365 40288416 62860940 84798273 115268894 722471 4378367 40288418 62860955 84798275 115268896 722473 4378369 40388582 62860957 84798277 115268898 722475 4378371 40388585 62860959 84798279 115268900 722477 4378373 40388592 62860961 84798295 115268902 722479 4378375 40388599 62860963 84798309 115268904 722483 4378377 40647131 62860965 84798321 115268906 722485 4378379 40784425 62860981 84798323 115270875 722487 4378383 40784429 62860983 84798325 115270877 722489 4378385 40795876 62860987 84798327 116543556 722493 4378387 42541061 62860989 84798343 116543560 722495 4378389 42541069 62860991 84798345 116543564 722497 4378391 42794782 62860994 84798347 116546686 722503 4378393 42794786 62860996 84798349 116546688 722505 4378395 44829186 62861000 84798351 116551153 722511 4378397 45111420 62861002 84798364 116551156 722513 4378399 45386482 62861004 84798366 116551162 722515 4378401 46016047 62861012 84798370 116551171 722521 4558868 46093898 62861015 84798372 116551175 722523 4680172 46093902 62861017 84798374 116551179 722525 4759539 46093906 62861019 84798377 116551183 722529 4759543 46093910 62861022 84798381 116551188 722531 4759547 46575858 62861024 84798383 116551192 722535 4759551 47078185 62861029 84798386 116551201 722537 4759555 47154907 62861031 84798388 116551207 722539 4759563 47154909 62861037 84798390 116551216 722541 4759567 47154911 62861041 84798397 116551226 722543 4759575 47154913 62861045 84798407 116551231 722545 4759579 47154915 62861054 85632219 116551235 722549 4759583 47154917 62868475 85642735 116551239 722553 4759587 47154919 62868477 85644222 116551244 722555 4759591 47154921 62868479 85644224 116551249 722557 4759595 47271269 62999493 85644226 116551258 722559 4759599 47271271 63102866 85644228 116551313 722561 4761194 47271273 63102872 85644230 116551317 722569 4761281 47271275 63102874 85644232 116551321 722571 4761283 47271277 63102876 85644600 116551325 722573 4837686 47271279 63102880 85644602 116551329 722581 4837688 47271281 63102882 85644604 116551333 722585 4837690 47271283 63102888 85650161 116551337 722587 4837692 47271285 63102892 85650163 116551341 722591 4837694 47271287 63102898 85650165 116551347 722593 4837696 47271289 63102900 85650167 116551351 722599 4837698 47271291 63102902 85650169 116551369 722601 5006350 47271295 63102904 85650171 116551373 722603 5006354 47271297 63102906 85650173 116551377 722605 5006356 47271299 63102908 85650175 116551381 722607 5006358 47271307 63102910 85650177 116551404 722609 5006360 47271309 63102912 85650179 116551413 722615 5019510 47271311 63102916 85650276 116551418 732737 5019512 47271313 63102920 85650278 116551422 732739 5019514 47271315 63102922 85650280 116551427 732741 5019522 47271317 63102924 85657010 116551431 732743 5019524 49073024 63102928 85658337 116551436 732745 5019526 49073036 63102938 85658632 116551446 732747 5019538 50199324 63102940 85660488 116551452 758588 5081714 50199334 63102942 85660492 116551772 758598 5081716 50831237 63102954 85660494 116551776 758600 5081718 50844518 63102962 85660497 116551780 762823 5081720 50844522 63102964 85660498 116551785 773589 5081722 50844526 63102966 85660502 116551790 790442 5102680 50844536 63102968 86439043 116553242 790450 5419682 50844540 63102970 86439047 116555276 790794 5419684 50844548 63102972 86439051 116555819 790802 5419700 50844552 63102974 86439053 116555821 790810 5419702 50871685 63102976 86439057 116555823 791015 5419704 50871687 63102980 86439061 116559889 791019 5419706 50898144 63102986 86439063 116560960 791023 5419708 50898148 63102988 86439071 116634471 791027 5419710 50898150 63102992 86439075 116634475 791031 5419712 50898152 63102994 86439081 116795086 791035 5419731 50898154 63102996 86439147 117576090 809552 5419738 50898158 63102998 86439151 118143176 809553 5419740 50898160 63103012 86439153 118143178 809554 5524134 50898162 63103014 87298995 118147088 845515 5524140 50898164 63103030 87298999 118147090 845517 5524142 50898170 63103032 87299001 118147092 845519 5524144 51103388 63103034 87299003 118147094 845521 5524146 51103390 63103040 87299007 118147096 845523 5524148 51103392 63103044 87299009 118147098 845525 5524150 51103394 63103046 87299011 118147100 845527 5566507 51103396 63103048 87299015 118147102 845529 5578779 51103398 63103054 88496317 118147104 845531 5578781 51103400 63103056 88496922 118147106 845533 5578783 51103402 63103070 90092372 118147108 845535 5578785 51103404 63103072 90092373 118147110 854111 5578787 51103406 63103076 90092374 118147112 871275 5578789 51103408 63103078 90092387 118147114 871819 5578791 51103410 63103086 90092910 118147116 871823 5578793 51103412 63103096 90092911 118147118 882261 5578795 51103414 63103098 90092912 118147120 882263 5578797 51103416 63103106 90092913 118147122 882265 5578799 51103418 63103108 90823178 118147125 882267 5578801 51103420 63103110 90823182 118147127 882269 5578803 51103422 63103112 90823186 118425771 882271 5578805 51103424 63103114 90823190 118425773 882273 5578807 51103522 63103116 90823196 118425775 882275 5578809 51103526 63103118 90823198 118490144 882277 5578811 51103528 63103120 90994745 118490148 882279 5578815 51103532 63103140 90994747 118490152 882281 5690395 51103534 63103142 90994751 118490156 882283 5690399 51103536 63103144 92115496 119359417 882285 5690403 51103538 63103146 92115497 119836694 882287 5709454 51103540 63103148 92130102 119836767 882289 5731228 51103542 63103150 92130103 119838997 882291 5731232 51103544 63103154 92131782 119839065 882293 5731236 51103546 63103156 92131783 119839355 882295 5731242 51103548 66096574 92131784 119839523 882297 5731252 51103550 66096603 92131785 119841342 882299 5921608 51103552 66096637 92133663 119841388 882301 5921610 51103554 66711101 92133665 119841425 882303 5921614 51103556 66711102 92137567 119841512 882305 5921618 51103558 66711103 92140334 121309186 882307 5921620 51103560 66711104 92140336 124042790 882309 5921622 51103562 66711105 92141530 124042792 882311 5921624 51103564 66711106 92155949 124042815 882313 5921626 51103566 66711107 92157443 126146964 882315 5921640 51103568 66711108 92157445 126146965 882317 6110569 51103570 66711109 92157453 126146966 882319 6179861 51851021 66711110 92157459 126147776 882321 6179863 51949938 66711111 92157461 126147812 882323 6179865 53988135 66711112 92158828 126147817 882325 6179867 53988137 66711114 92158980 126147952 882327 6179869 54034484 66711116 92161545 126147954 882329 6492198 54145422 66711117 92249233 126147956 882331 6492200 54145426 66711118 92298212 126152193 894090 6492202 54145440 66711119 92298539 126152196 904629 6492204 54781098 66711120 92315622 126633956 913352 6648587 54781100 66711123 92315624 126633957 929640 6649889 54781102 66711124 92315626 126633958 929642 6649895 54781104 66711125 92315628 134125852 944925 6708204 54781106 66711126 92332837 134125853 950049 7012704 54781108 66711128 92332841 134125854 973411 7012706 54781110 66711129 92348102 134128019 973415 7024356 54781112 66711130 92348670 134269772 999107 7160978 54781126 66711131 92349881 134273023 1020008 7673384 54781129 66711132 92360819 145850477 1020012 7673388 54781202 66711133 92370888 145850518 1020016 7673392 54781204 66711134 92381676 145850519 1070309 7745134 54781206 66711135 92496960 145850520 1070313 8250280 54781208 66711136 92520581 145850521 1070315 8777870 54781213 66711137 92520583 145850522 1070317 8777874 54781216 66711138 92520584 145850523 1070321 8777878 54781218 66711139 92520586 145850524 1070325 8777880 54781220 66711140 92575636 145850525 1070327 8777884 54781223 66711141 92589636 145850526 1070347 8777888 54781225 66711142 92589637 145850527 1136554 8777890 54781227 66711143 92589638 145850528 1136556 8777892 54781229 66711144 92589639 145850529 1208913 9295278 54781231 66711145 92589640 145850530 1235764 9295280 55274149 66711146 92589641 145850531 1235766 9295282 55274153 66711147 92589642 145850532 1235768 9295284 55274159 67509857 92589643 145850533 1235770 9295286 55274163 67509861 92589644 145850534 1235772 9295290 55824376 68148126 92589645 145850535 1235774 9295292 56118076 68148140 92589646 145850536 1245380 9295296 56118080 68148142 92589647 145850537 1245382 9295298 56292538 68148144 92589648 145850558 1255605 9295300 56294837 68148150 92589649 145850561 1255607 9437312 56294841 68148152 92589650 145850563 1255608 9927567 56399565 68148154 92589651 145854440 1255609 9928208 56609227 68148158 92589652 145856824 1255612 9968441 56609228 68148160 92589653 145859735 1292860 9968443 56609229 68148164 92589656 148355517 1292862 9968486 56609230 68148166 92600475 148355518 1353813 9968488 56609232 68148174 92600479 148355519 1353815 9968490 56609235 70797818 92600487 148355520 1353817 9968492 56742105 70797820 92607622 148355521 1353819 9968494 56742106 70797822 92667306 148355522 1353821 9968496 58003567 70797824 92667307 148355523 1353825 9968498 58003568 70797826 92667308 148355524 1353827 9968500 58003569 70797828 92667309 148355525 1353831 9997457 58003570 70797830 92667310 148355526 1370131 10636524 58003571 70797832 92667329 148355527 1370135 11229436 58003572 70797834 92667331 148355528 1370137 11343336 58003573 70797836 92798195 148355529 1495627 11343337 58003587 70797838 92798196 148355530 1495628 11876718 58003588 70797842 92798197 148355531 1495629 11876734 58003589 70797844 92798198 148355532 1495630 11876735 58003608 70797846 92798199 148355533 1495631 11876736 58003609 70797850 92798218 148540957 1495632 11876737 58003610 70797852 92798220 148578450 1495633 11876738 58003611 70797854 92824835 148578452 1495634 11876739 58003612 70797856 92834676 148578454 1495635 11876740 58003613 70797858 92835832 148578455 1495637 11876741 58003614 70797860 92835834 148578456 1495638 11878173 58003615 70797866 92835836 148578457 1495639 11878175 58003616 70797870 92839400 148578458 1495640 11878177 58003618 70797872 92839402 148578460 1495641 11992075 58003619 70797874 92839403 149849068 1495642 11992193 58003620 70797876 92839404 149849080 1495643 12003249 58003622 70797878 92839405 149849084 1495644 12003251 58003623 70797884 92839406 149849088 1495645 12003253 58003624 70797886 92839407 150447881 1495646 12003255 58003625 70797888 92839408 150447883 1495647 12003257 58003626 70797890 92839409 150447885 1495648 12655491 58003627 70797894 92845038 150447887 1495649 12655493 58003628 70797898 92845490 150450134 1495650 12655500 58003629 70798601 92845651 150450135 1495651 12655502 58003630 70798603 92855396 150450136 1495652 12655504 58003631 70798605 92855400 150450137 1532001 12655519 58003632 70798607 92855404 150450138 1532002 12655521 58003633 70798609 92855408 150450139 1532027 12655525 58003634 70798611 92855412 150450140 1552277 12655527 58003656 70798613 92855416 150450636 1552283 12655529 58003657 70798615 92855420 150453145 1552285 12655531 58003658 70798617 92855424 150453147 1552287 12655541 58003659 70798619 92855428 150453149 1552291 12655558 58003660 70798621 92855432 150453151 1552295 12655565 58003661 70798623 92855436 150453153 1552299 12655567 58032596 70798627 92855441 150453154 1552319 12655569 58032603 70798629 92855444 150453155 1561601 12655643 58032606 70798631 92856854 150453156 1561605 12655655 58194104 70798633 92856855 150453157 1561607 12655662 58194120 70798635 92856859 150453159 1561609 12655665 58194136 70798637 92857001 150453161 1561611 12655672 58202701 70798639 92857003 150453163 1572702 12655713 58202709 70798641 92857012 150453165 1572704 12655723 58202711 70798643 92857016 150453167 1572706 12655730 58202713 70798645 92857018 150453169 1572708 12655732 58202715 70798649 92858156 150453171 1572710 12655736 58202717 70798653 92861312 150453174 1657324 12655738 58202719 70798655 92861313 150453213 1657326 12655740 58202721 70798657 92861314 150453216 1657328 12655748 58202723 70798659 92862784 153590356 1673592 12655751 58202725 70798661 92875826 153590359 1673602 12710669 58202727 70798667 92878541 153590361 1710418 12710671 58202729 70798669 92878543 153590363 1770403 12734084 58202733 70798671 92878545 153590365 1770415 12734089 58202735 70798673 92903931 153590367 1773056 12750933 58202737 70798675 92905358 153590371 1778125 12836990 58222454 70798677 92905360 156149223 1785869 12957385 58222456 70798679 92905362 156149224 1785873 12957387 58222458 70798681 94034254 156149225 1785877 13170940 58222460 70798683 94034257 156229617 1800286 13170944 58222462 70798685 94034261 156557387 1813653 13170948 58222464 70798687 94034264 156557389 1813655 13171333 58222466 70798690 94034267 156557391 1813657 13171339 58222468 70798692 94034271 156557393 1834498 13171341 58222470 70798694 94034285 156557399 1834563 13171343 58222473 70798696 94034316 156557403 1834564 13447996 58222476 70798698 94034339 156557405 1835872 13448000 58222478 70798700 94034342 156557407 1835873 13448002 58222480 70798702 94034384 156557411 1839291 13448004 58222482 70798706 94034387 156562058 1864110 13448006 58222484 70798708 94034390 157087534 1864112 13448010 58222487 70798710 94034393 157896695 1864114 13448012 58222489 70798712 94035272 157896697 1864116 13448016 58222491 70798716 94035284 157903220 1864118 13448018 58222493 70798718 94035289 158055245 1864136 13448022 58222497 70798720 94035298 158055254 1864138 13549147 58222499 70798722 94035300 158055268 1890131 13785652 58222501 70798724 94035312 158055282 1890133 13939245 58222503 70798732 94469910 158055285 1905798 13939277 58222505 70798734 94469912 158055288 1905937 13939331 58222507 70798736 94469914 158058441 1905941 13991697 58222509 70798738 94469922 158731523 1911732 14150696 58222511 70798742 94469924 158731524 1922370 14150698 58222513 70798744 94469926 158731525 1922438 14290262 58222515 70798750 95007504 158731526 1922466 14573212 58222517 70798752 95007510 158731527 1922501 14573214 58222519 70798758 95007512 158731528 1922528 14573216 58222521 70798760 95007514 158731529 1922535 14573218 58222523 70798764 95007516 158731530 1922602 14573220 58222525 70798766 95007518 158731531 1922618 14573222 58222527 70798768 95007520 158731532 1922645 14573226 58222529 70798770 95007522 158731533 1922679 14573254 58222531 70798772 95007524 158731534 1922796 14573256 58222533 70798774 95007526 158731536 1922805 14573258 58222535 70798776 95007528 158731538 1932772 14573260 58222537 70798778 95007530 158731539 1943727 14573262 58222539 70798780 95007532 158731540 2058533 14573264 58222541 70798782 95007534 158731541 2058535 14573266 58222543 70798784 95007536 158731542 2058678 14573268 58222545 70798786 95007538 158731545 2072271 14573270 58222547 70798788 95007540 158731546 2072273 14573272 58222549 70798792 95007542 158731547 2072279 14573274 58222551 70798794 95007544 158731548 2072981 14573276 58222553 70798796 95101759 158731550 2078359 14573278 58222556 70798798 95101761 158731551 2078371 14588864 58222558 70798800 95101767 158731552 2078373 14588866 58222560 70798802 95101769 158731553 2169989 14588868 58222562 70798804 95101777 158731554 2169990 14588870 58222564 70798806 98956195 158731555 2172285 14588872 58222566 70798808 98956209 158731556 2173403 14597098 58222568 70798810 98956219 158731557 2175768 14597112 58222570 70798812 98956223 158731558 2175852 14597124 58222572 70798814 98956232 158731559 2175867 14597127 58222575 70798816 98956244 158731560 2218123 14625743 58222577 70798818 98956249 158731561 2239113 14625918 58222579 70798820 98956255 158731562 2239115 14626493 58222581 70798824 98956261 158731563 2253439 14716957 58222583 70798826 98956263 158731564 2266632 14716961 58222585 70798828 98956271 158731565 2266634 14716969 58222587 70798830 98956277 158731566 2291087 14716971 58222589 70798832 98956279 158731567 2293965 14716973 58222591 70798834 98956281 158731568 2293967 15011457 58222593 70798836 98956285 158731569 2306827 15099974 58222595 70798838 98956289 158744132 2306829 15277619 58222597 70798840 98956291 158744140 2345025 15419020 58222599 70798842 98956293 158744148 2345029 15859220 58222601 70798844 98956299 158744156 2345031 15986229 58222603 70798846 98956301 158744164 2345033 16508167 58222605 70798848 98956303 158746355 2385484 16554974 58222607 70798850 98956305 158746363 2385486 16923186 58222609 70798852 98956307 158746371

APPENDIX B GI Numbers of Lambda Light Chains Used to Derive the Vλ Libraries 31454 3142529 4566076 9968397 51103608 77379760 32808 3142531 4566078 9968401 51103612 77379824 32812 3142533 4566082 9968403 51103614 77379826 33335 3142535 4566084 9968405 51103616 77379828 33368 3142537 4566086 9968409 51490956 77379830 33383 3142539 4566088 9968411 54781261 77379832 33387 3142541 4566090 9968413 61815560 77379834 33412 3142543 4566092 9968415 62720404 77379836 33429 3142545 4566094 9968417 62720406 77379838 33431 3142547 4566096 9968419 62720408 77379840 33433 3142549 4566098 9968421 62720412 77379842 33703 3142553 4566101 9968423 62860947 77379846 33711 3142556 4566105 9968425 62860950 77379848 37918 3142558 4732059 9968427 62860967 77379850 37920 3142562 4761253 9968429 62860969 77379855 37922 3142564 4761255 9968433 62860971 77379857 37923 3142566 4761257 9968435 62860973 77379859 38359 3142569 4761259 9968437 62860975 77379861 38360 3142573 4761261 9968439 62860977 77379863 38364 3142577 4761263 10636511 62860979 77379865 38365 3142579 4761265 10636514 62860985 77379867 38366 3142581 4761267 10636518 62861006 77379869 38368 3142583 4761269 10636521 62861008 77379871 186078 3142585 4761271 10636527 62861010 77379875 186080 3142587 4761273 11992185 62861047 77379877 186082 3142589 4761277 11992187 62999489 77379879 186084 3142591 4761279 11992189 62999497 77379882 186086 3142593 4927957 11992191 62999501 77379884 186088 3142595 5019504 11992195 62999509 77379886 186090 3142597 5019506 11992197 70888031 77379888 186092 3142599 5019516 11992199 70888035 77379890 186094 3142601 5019518 11992201 70888037 77379894 186096 3142603 5019520 12666922 70888041 77379896 186097 3142612 5019528 12666924 70888043 77379900 186111 3142614 5019530 12666926 70888045 77379908 186162 3142616 5019532 12666928 70888047 77379910 186164 3142618 5019534 12666930 70888049 77379912 186168 3142620 5019536 12666932 70888051 77379916 186170 3142649 5174362 12666934 70888053 77379918 186172 3142651 5174364 12666936 70888055 80975584 186175 3142653 5174366 12666938 70888057 80975588 298556 3142656 5174378 12666940 70888059 80975598 405223 3142658 5524086 12666942 70888061 80975622 405227 3142660 5524106 12666944 70888063 80975628 409040 3142662 5524108 12666946 70888065 80975632 409041 3142668 5524118 12666948 70888067 80975636 409043 3142670 5524122 12666952 70888069 81020028 433485 3142672 5524132 12666954 70888071 81020064 434041 3142674 5578817 12666956 70888073 86438995 434045 3142676 5578819 12666958 70888075 86439001 439514 3142678 5578823 12666960 70888077 86439005 439516 3142680 5578825 12666962 70888079 86439015 441251 3142684 5578827 12830380 70888081 86439017 460854 3153359 5578829 12830382 70888083 86439087 460856 3153361 5578831 12830384 70888085 86439089 460860 3153365 5578833 13276707 70888087 86439091 465157 3153366 5911837 13877276 70888089 86439093 465167 3153368 6492194 14279402 70888091 86439095 465171 3153374 6492196 14279404 70888093 86439097 465175 3153376 6492206 14279406 70888095 86439099 469249 3335577 6492208 17226627 70888097 86439101 483911 3335579 6492210 17226649 70888099 86439105 487824 3335585 6492212 18307305 70888103 86439127 487825 3335587 6643078 18307307 70888105 86439133 487828 3335591 6643082 18307309 70888109 86439137 493153 3388046 6643086 18307311 70888111 86439139 506426 3388048 6643088 18307313 70888113 86439141 506428 3388050 6643090 18307315 70888115 90994749 515765 3388054 6643098 18307317 70888117 95007506 532599 3388056 6643104 18307319 70888121 95007546 532600 3388058 6643106 18307321 70888123 95007548 532603 3388060 6643114 18307329 70888125 95007550 560845 3388062 6643118 21311290 70888127 95007552 575230 3388064 6643120 21311292 70888129 95007554 575238 3388066 6643124 21669150 70888133 95007556 575242 3388070 6643126 21669152 70888137 95007558 685021 3388072 6643128 21669154 70888139 95007560 773591 3388074 6643136 21669156 70888141 95007562 871362 3388080 6643138 21669158 70888143 95007564 987068 3747019 6643154 21669160 70888147 95007566 987076 3821077 6643156 21669162 70888149 95007570 998390 3821078 6643158 21669164 70888151 95007572 998394 3821079 6643162 21669166 70888155 95007576 1055278 3821080 6643168 21669172 70888157 95007578 1070329 3821081 6643170 21669174 70888159 109240683 1070341 3821082 6643172 21669176 70888161 109240697 1070349 3821083 6643176 21669178 70888163 109240743 1143195 3821084 6643178 21669180 70888165 109240749 1200068 3821086 6643180 21669182 70888167 109240754 1235776 3821087 6643182 21669184 70888169 109240756 1235778 3821089 6643184 21669186 70888171 109240758 1235780 3821090 6643186 21669188 70888173 116795127 1235782 3821091 6643188 21669190 70888179 116795192 1255606 3821092 6643192 21669192 70888181 146336934 1255610 3821093 6643196 21669194 70888183 156632919 1255611 3821094 6643198 21669196 70888185 156632943 1255613 3821095 6643200 21669198 70888187 156632945 1552313 3821096 6643202 21669200 70888193 156632975 1561599 3821097 6643204 21669204 70888195 156633095 1770407 4103646 6643210 21669206 70888197 156633103 1864134 4103648 6643214 21669210 70888199 156633141 1864140 4103650 6643218 21669212 70888201 156633153 1864142 4103652 6643220 21669214 70888204 156633155 1864144 4103654 6643224 21669218 70888206 156633159 2078365 4103656 6643226 21669220 70888208 156633171 2654039 4103658 6643230 21669222 70888210 156633179 2654043 4103660 6643232 21669224 70888212 156633199 2865485 4103672 6643238 21669226 70888216 156633203 3023094 4324023 6643240 21669228 70888218 156633209 3023096 4324025 6643242 21669230 70888220 156633211 3023098 4324029 6643244 21669232 70888222 156633225 3023100 4324031 6643248 21669234 70888224 156633229 3023102 4324037 6643250 21669236 70888228 156633237 3023104 4324039 6643254 21669238 70888230 156633241 3023106 4324043 6643256 21669240 70888232 156633245 3023108 4324047 6643258 21669242 70888234 156633253 3023110 4324055 6643268 21669244 70888236 156633255 3023112 4324057 6643272 21669248 70888238 156633267 3023114 4324061 6643274 21669252 70888240 156633283 3023116 4324063 6643276 21669254 70888242 157093725 3023118 4324067 6643278 21669256 70888244 170684323 3023120 4324069 6643280 21669260 70888246 170684325 3023122 4324073 6643282 21669262 70888248 170684329 3023126 4324075 6643286 21669264 70888250 170684331 3023130 4324077 6643290 21669266 70888252 170684333 3023132 4324085 6643292 21669268 70888254 170684335 3091153 4324087 6643294 21669270 70888258 170684339 3091155 4324089 6643296 21669272 70888260 170684341 3091157 4324091 6643302 21669274 70888262 170684345 3091159 4324093 6643304 21669276 70888264 170684349 3091161 4324097 6643308 21669278 70888266 170684351 3091163 4324103 6643314 21669280 70888268 170684355 3091165 4324107 6643318 21669288 70888270 170684363 3091167 4324111 6643328 21998780 70888272 170684365 3091169 4324113 6643344 21998782 70888274 170684369 3091171 4324115 6643352 21998784 70888276 170684371 3091173 4324117 6643354 21998786 70888278 170684373 3091175 4324123 6643358 21998792 70888280 170684375 3091177 4324125 6643360 21998794 70888282 170684379 3091179 4324127 6643362 21998800 70888284 170684381 3091181 4324139 6643366 21998802 70888286 170684385 3091183 4324145 6643368 21998804 70888288 170684387 3091185 4324151 6643374 23194484 70888290 170684389 3091187 4324155 6643376 23194488 70888292 170684397 3091191 4324157 6643378 23194492 70888294 170684405 3091193 4324159 6643382 23194496 70888296 170684407 3091195 4324163 6643386 23343556 70888304 170684409 3091197 4324169 6643390 24474079 70888306 170684411 3091201 4324175 6643392 27369031 71482628 170684417 3091203 4324177 6643402 27369033 71482632 170684419 3091205 4324181 6643416 27369035 77378177 170684423 3091207 4324187 6643418 27369037 77378188 170684425 3091209 4324189 6643424 27369045 77378257 170684427 3091213 4324193 6643428 27369047 77378266 170684429 3093861 4324197 6643436 27369051 77378268 170684431 3093863 4324199 6643446 27369053 77378270 170684433 3093865 4324205 6643448 27369058 77378273 170684439 3093867 4324207 6643450 27369060 77378277 170684443 3093869 4324209 6643452 27369064 77378280 170684449 3093871 4324211 6643456 27369068 77378282 170684451 3093873 4324213 6643470 27369075 77378284 170684453 3093875 4324215 6643474 27369082 77378286 170684461 3093877 4324221 6643478 27369084 77378288 170684469 3093879 4324223 6643484 27369088 77378291 170684473 3093881 4324229 6643488 27818828 77378293 170684489 3093883 4324231 6643492 28394695 77378298 170684495 3093885 4324245 6643500 28394699 77378300 170684497 3093887 4324247 6643512 28394703 77378303 170684499 3093889 4324249 6643514 28394707 77378305 170684501 3093891 4324251 6643528 28394711 77378307 170684507 3093895 4324255 6643534 28394715 77378309 170684513 3093903 4324257 6643558 28848877 77378312 170684515 3142451 4324261 6643560 28848881 77378316 170684517 3142453 4324263 6643562 28848885 77378318 170684527 3142455 4324265 6643564 29342115 77378320 170684531 3142457 4324271 6643572 33304654 77378322 170684535 3142459 4324273 6643574 40647151 77378377 170684537 3142461 4324275 6643580 47271301 77378379 170684539 3142465 4324283 6643582 47271303 77378381 170684541 3142467 4324285 6643584 47271319 77378383 170684545 3142471 4468355 6643586 47271321 77378385 170684549 3142475 4468367 6643588 47271323 77378387 170684553 3142477 4468369 6643592 47271325 77378389 170684555 3142479 4468371 6643596 50199320 77378392 170684557 3142481 4565964 6643598 50199322 77378394 170684561 3142483 4565966 6643600 50199328 77378396 170684565 3142485 4565996 6643602 50199330 77378398 170684567 3142487 4566007 6643604 50199338 77378400 170684569 3142489 4566009 6643606 50199340 77378402 170684571 3142491 4566016 6643614 50871689 77379590 170684583 3142493 4566021 6643628 51103426 77379620 170684589 3142495 4566023 6643630 51103428 77379622 170684591 3142497 4566025 6649891 51103430 77379624 170684593 3142499 4566029 6649893 51103434 77379632 170684597 3142503 4566045 8920222 51103436 77379642 170684599 3142505 4566049 8920226 51103572 77379644 170684601 3142507 4566051 9864840 51103574 77379646 170684603 3142509 4566053 9968383 51103576 77379675 170684607 3142511 4566055 9968385 51103588 77379677 170684609 3142515 4566057 9968387 51103590 77379726 170684613 3142517 4566059 9968389 51103592 77379728 170684617 3142519 4566061 9968391 51103600 77379730 170684619 3142521 4566065 9968393 51103602 77379738 3142527 4566074 9968395 51103606 77379740 

1. A library of synthetic polynucleotides, wherein said polynucleotides encode at least 10⁶ unique antibody CDRH3 amino acid sequences comprising: (i) an N1 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N1 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by B cells; (ii) a non-human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (iii) an N2 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N2 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N2 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by B cells; and (iv) a human CDRH3H3-JH amino acid sequence, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them.
 2. The library of claim 1, wherein the B cells are human B cells.
 3. The library of claim 1, wherein the B cells are non-human B cells.
 4. The library of claim 1, wherein the B cells are vertebrate B cells.
 5. The library of claim 1, wherein the non-human CDRH3 DH amino acid sequence is a sequence from a vertebrate species.
 6. The library of claim 5, wherein the vertebrate species is selected from the group consisting of Mus musculus, Camelus sp., Llama sp., Camelidae sp., Raja sp., Ginglymostoma sp., Carcharhinus sp., Heterodontus sp., Hydrolagus sp., Ictalurus sp., Gallus sp., Bos sp., Marmaronetta sp., Aythya sp., Netta sp., Equus sp., Pentalagus sp., Bunolagus sp., Nesolagus sp., Romerolagus sp., Brachylagus sp., Sylvilagus sp., Oryctolagus, sp., Poelagus sp., Ovis sp., Sus sp., Gadus sp., Salmo sp., Oncorhynchus sp, Macaca sp., Rattus sp., Pan sp., Hexanchus sp., Heptranchias sp., Notorynchus sp., Chlamydoselachus sp., Heterodontus sp. Pristiophorus sp., Pliotrema sp., Squatina sp., Carcharia sp., Mitsukurina sp., Lamma sp., Isurus sp., Carcharodon sp., Cetorhinus sp., Alopias sp., Nebrius sp., Stegostoma sp., Orectolobus sp., Eucrossorhinus sp., Sutorectus sp., Chiloscyllium sp., Hemiscyllium sp., Brachaelurus sp., Heteroscyllium sp., Cirrhoscyllium sp., Parascyllium sp., Rhincodon sp., Apristurus sp., Atelomycterus sp., Cephaloscyllium sp., Cephalurus sp., Dichichthys sp., Galeus sp., Halaelurus sp., Haploblepharus sp., Parmaturus sp., Pentanchus sp., Poroderna sp., Schroederichthys sp., Scyliorhinus sp., Pseudotriakis sp., Scylliogaleus sp., Furgaleus sp., Hemitriakis sp., Mustelus sp., Triakis sp., Iago sp., Galeorhinus sp., Hypogaleus sp., Chaenogaleus sp., Hemigaleus sp., Paragaleus sp., Galeocerdo sp., Prionace sp., Sciolodon sp., Loxodon sp., Rhizoprionodon sp., Aprionodon sp., Negaprion sp., Hypoprion sp., Carcharhinus sp., Isogomphodon sp., Triaenodon sp., Sphyrna sp., Echinorhinus sp., Oxynotus sp., Squalus sp., Centroscyllium sp., Etmopterus sp., Centrophorus sp., Cirrhigaleus sp., Deania sp., Centroscymnus sp., Scymnodon sp., Dalatias sp., Euprotomicrus sp., Isistius sp., Squaliolus sp., Heteroscymnoides sp., Somniosus sp. and Megachasma sp.
 7. An antibody isolated from the polypeptide expression products of the library of claim
 1. 8. The library of claim 1, wherein the CDRH3 amino acid sequences are expressed as part of full-length heavy chains.
 9. The library of claim 1, wherein the polynucleotides further encode an alternative scaffold.
 10. A library of polypeptides encoded by the synthetic polynucleotide library of claim
 1. 11. A library of vectors comprising the polynucleotide library of claim
 1. 12. A population of cells comprising the vectors of claim
 11. 13. The cells of claim 12, wherein the cells are yeast cells.
 14. The cells of claim 12, wherein the cells are S. cerevisiae.
 15. A method of preparing a synthetic polynucleotide library comprising providing the polynucleotide sequences of claim 1 and assembling said polynucleotide sequences in the order [N1]-[DH]-[N2]-[H3-JH].
 16. A method of isolating one or more host cells expressing one or more antibodies, the method comprising: (i) expressing a polypeptide comprising a CDRH3 sequence of claim 1 in one or more host cells; (ii) contacting the host cells with one or more antigens; and (iii) isolating one or more host cells having antibodies that bind to the one or more antigens.
 17. A kit comprising the library of synthetic polynucleotides of claim
 1. 18. The CDRH3 amino acid sequences encoded by the library of synthetic polynucleotides of claim 1 in computer readable form. 